Methods of using interleukin-3 (IL-3) mutant polypeptides for ex-vivo expansion of hematopoietic stem cells

ABSTRACT

The present invention relates to recombinant human interleukin-3 (hIL-3) variant or mutant proteins (muteins). These hIL-3 muteins contain amino acid substitutions and may also have amino acid deletions at both the N- and C-termini. The invention also relates to pharmaceutical compositions containing the hIL-3 muteins and methods for using them. Additionally, the present invention relates to recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 muteins, related microbial expression systems, and processes for making the hIL-3 muteins using the microbial expression systems. Included in the present invention are deletion mutants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus, and from 1 to 15 amino acids (a.a.119 to 133) have been deleted from the C-terminus, and which also contain amino acid substitutions in the polypeptide. These hIL-3 multiple mutation polypeptides may have biological activities similar to or better than hIL-3 and, in some cases, may also have an improved side effect profile.

This application is a continuation of U.S. application Ser. No. 08/411,796, filed Apr. 6, 1995, now U.S. Pat. No. 5,677,149; which entered the U.S. national stage under 35 USC §371 from PCT/US93/11198, filed Nov. 22, 1993; which is a continuation-in-part of U.S. application Ser. No. 07/981,044, filed Nov. 24, 1992, now abandoned.

This is a continuation-in-part of U.S. application Ser. No. 07/981,044 filed Nov. 24, 1992 which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to mutants or variants of human interleukin-3 (hIL-3) which contain one or more amino acid substitutions and which may have portions of the native hIL-3 molecule deleted. These hIL-3 single and multiple mutation polypeptides retain one or more activities of native hIL-3 and may also show improved hematopoietic cell-stimulating activity and/or an improved activity profile which may include reduction of undesirable biological activities associated with native hIL-3.

BACKGROUND OF THE INVENTION

Colony stimulating factors (CSFs) which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. CSFs in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.

Because of its ability to stimulate the proliferation of a number of different cell types and to support the growth and proliferation of progenitor cells, IL-3 has potential for therapeutic use in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.

Interleukin-3 (IL-3) is a hematopoietic growth factor which has the property of being able to promote the survival, growth and differentiation of hematopoietic cells. Among the biological properties of IL-3 are the ability (a) to support the growth and differentiation of progenitor cells committed to all, or virtually all, blood cell lineages; (b) to interact with early multipotential stem cells; (c) to sustain the growth of pluripotent precursor cells; (d) to stimulate proliferation of chronic myelogenous leukemia (CML) cells; (e) to stimulate proliferation of mast cells, eosinophils and basophils; (f) to stimulate DNA synthesis by human acute myelogenous leukemia (AML) cells; (g) to prime cells for production of leukotrienes and histamines; (h) to induce leukocyte chemotaxis; and (i) to induce cell surface molecules needed for leukocyte adhesion.

Mature human interleukin-3 (hIL-3) consists of 133 amino acids. It has one disulfide bridge and two potential glycosylation sites (Yang, et al., CELL 47:3 (1986)).

Murine IL-3 (mIL-3) was first identified by Ihle, et al., J. IMMUNOL. 126:2184 (1981) as a factor which induced expression of a T cell associated enzyme, 20-hydroxysteroid dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and lymphoid progenitor cells.

In 1984, cDNA clones coding for murine IL-3 were isolated (Fung, et al., NATURE 307:233 (1984) and Yokota, et al., PROC. NATL. ACAD. SCI. USA 81:1070 (1984)). The murine DNA sequence coded for a polypeptide of 166 amino acids including a putative signal peptide.

The gibbon IL-3 sequence was obtained using a gibbon cDNA expression library. The gibbon IL-3 sequence was then used as a probe against a human genomic library to obtain a human IL-3 sequence.

Gibbon and human genomic DNA homologues of the murine IL-3 sequence were disclosed by Yang, et al., CELL 47:3 (1986). The human sequence reported by Yang, et al. included a serine residue at position 8 of the mature protein sequence. Following this finding, others reported isolation of Pro⁸ hIL-3 cDNAs having proline at position 8 of the protein sequence. Thus it appears that there may be two allelic forms of hIL-3.

Dorssers, et al., GENE 55:115 (1987), found a clone from a human cDNA library which hybridized with mIL-3. This hybridization was the result of the high degree of homology between the 3′ noncoding regions of mIL-3 and hIL-3. This cDNA coded for an hIL-3 (Pro⁸) sequence.

U.S. Pat. Nos. 4,877,729 and 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.

Clark-Lewis, et al., SCIENCE 231:134 (1986) performed a functional analysis of murine IL-3 analogues synthesized with an automated peptide synthesizer. The authors concluded that the stable tertiary structure of the complete molecule was required for full activity. A study on the role of the disulfide bridges showed that replacement of all four cysteines by alanine gave a molecule with {fraction (1/5000)}th the activity as the native molecule. Replacement of two of the four Cys residues by Ala(Cys⁷⁹, Cys¹⁴⁰→Ala⁷⁹, Ala¹⁴⁰) resulted in an increased activity. The authors concluded that in murine IL-3 a single disulfide bridge is required between cysteines 17 and 80 to get biological activity that approximates physiological levels and that this structure probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function. (Clark-Lewis, et al., PROC. NATL. ACAD. SCI. USA 85:7897 (1988)).

International Patent Application (PCT) WO 89/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser⁸→Pro⁸ replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.

EP-A-0275598 (WO 88/04691) illustrates that Ala¹ can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, e.g., two double mutants, Ala¹→Asp¹, Trp¹³→Arg¹³ (pGB/IL-302) and Ala¹→Asp¹, Met³→Thr³ (pGB/IL-304) and one triple mutant Ala¹→Asp¹, Leu⁹→Pro⁹, Trp¹³→Arg¹³ (pGB/IL-303).

WO 88/05469 describes how deglycosylation mutants can be obtained and suggests mutants of Arg⁵⁴Arg⁵⁵ and Arg¹⁰⁸Arg¹⁰⁹Lys¹⁰ might avoid proteolysis upon expression in Saccharomyces cerevisiae by KEX2 protease. No mutated proteins are disclosed. Glycosylation and the KEX2 protease activity are only important, in this context, upon expression in yeast.

WO 88/06161 mentions various mutants which theoretically may be conformationally and antigenically neutral. The only actually performed mutations are Met²→Ile² and Ile¹³¹→Leu¹³¹. It is not disclosed whether the contemplated neutralities were obtained for these two mutations.

WO 91/00350 discloses nonglycosylated hIL-3 analog proteins, for example, hIL-3 (Pro⁸Asp¹⁵Asp⁷⁰), Met³ rhul-3 (Pro⁸Asp¹⁵Asp⁷⁰); Thr⁴ rhuL-3 (Pro⁸Asp¹⁵Asp⁷⁰) and Thr⁶ rhuIL-3 (Pro⁸Asp¹⁵Asp⁷⁰). It is said that these protein compositions do not exhibit certain adverse side effects associated with native hIL-3 such as urticaria resulting from infiltration of mast cells and lymphocytes into the dermis. The disclosed analog hIL-3 proteins may have N termini at Met³, Thr⁴, or Thr⁶.

WO 90/12874 discloses cysteine added variants (CAVs) of IL-3 which have at least one Cys residue substituted for a naturally occurring amino acid residue.

SUMMARY OF THE INVENTION

The present invention relates to recombinant human interleukin-3 (hIL-3) variant or mutant proteins (muteins). These hIL-3 muteins contain amino acid substitutions and may also have amino acid deletions at either/or both the N- and C-termini. Preferably, these mutant polypeptides of the present invention contain one to three amino acids which differ from the amino acids found at the corresponding positions in the native hIL-3 polypeptide. The invention also relates to pharmaceutical compositions containing the hIL-3 muteins, DNA coding for the muteins, and methods for using the muteins. Additionally, the present invention relates to recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 muteins, related microbial expression systems, and processes for making the hIL-3 muteins using the microbial expression systems.

The present invention includes mutants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus, and in which from one to three amino acid substitutions have been made. Preferred muteins of the present invention are those in which amino acids 1 to 14 have been deleted from the N-terminus, or amino acids 126 to 133 have been deleted from the C-terminus, and which both also contain from one to three amino acid substitutions in the polypeptide sequence. These hIL-3 multiple mutation polypeptides may have biological activities similar to or better than hIL-3 and, in some cases, may also have an improved side effect profile, i.e., some muteins may have a better therapeutic index than native hIL-3. The present invention also provides muteins which may function as IL-3 antagonists or as discrete antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols. In addition to the use of the hIL-3 mutant polypeptides of the present invention in vivo, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.

Antagonists of hIL-3 would be particularly useful in blocking the growth of certain cancer cells like AML, CML and certain types of B lymphoid cancers. Other conditions where antagonists would be useful include those in which certain blood cells are produced at abnormally high numbers or are being activated by endogenous ligands. Antagonists would effectively compete for ligands, presumably naturally occurring hemopoietins including and not limited to IL-3, GM-CSF and IL-5, which might trigger or augment the growth of cancer cells by virtue of their ability to bind to the IL-3 receptor complex while intrinsic activation properties of the ligand are diminished. IL-3, GM-CSF and or IL-5 also play a role in certain asthmatic responses. An antagonist of the IL-3 receptor may have utility in this disease by blocking receptor-mediated activation and recruitment of inflammatory cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the human IL-3 gene for E. coli expression (pMON5873), encoding the polypeptide sequence of natural (wild type) human IL-3 [SEQ ID NO:128], plus an initiator methionine, as expressed in E. coli, with the amino acids numbered from the N-terminus of the natural hIL-3.

FIG. 2: ClaI to NsiI Replacement Fragment. FIG. 2 shows the nucleotide sequence of the replacement fragment used between the ClaI and NsiI sites of the hIL-3 gene. The codon choice used in the fragment corresponds to that found in highly expressed E. coli genes (Gouy and Gautier, 1982). Three new unique restriction sites, EcoRV, XhoI and PstI were introduced for the purpose of inserting synthetic gene fragments. The portion of the coding sequence shown encodes hIL-3 amino acids 20-70.

FIGS. 3A and 3B shows the nucleotide and amino acid sequence of the gene in pMON5873 with the sequence extending from NcoI through HindIII. The codon choices used to encode amino acids 1-14 and 107-133 correspond to that found in highly expressed E. coli genes.

FIG. 4 shows the construction of the plasmid vector pMON5846 which encodes [Met-(1-133) hIL-3 (Arg¹²⁹)].

FIG. 5 shows the construction of the plasmid vector pMON5847 (ATCC 68912) which encodes [Met-(1-133) hIL-3 (Arg¹²⁹)].

FIG. 6 shows the construction of plasmid vector pMON5853 which encodes [Met-(15-133) hIL-3 (Arg¹²⁹)].

FIG. 7 shows the construction of the plasmid vector pMON5854 which encodes [Met-(1-133) hIL-3 (Arg¹²⁹)].

FIG. 8 shows the DNA sequence and resulting amino acid sequence of the lamB signal peptide.

FIG. 9 shows the construction of the plasmid vector pMON5978 which encodes Met-Ala-(15-125) hIL-3.

FIG. 10 shows the construction of the plasmid vector pMON5988 which encodes Met-Ala(15-125) hIL-3.

FIG. 11 shows the construction of the plasmid vector pMON5887 which encodes Met-(1-125) hIL-3.

FIG. 12 shows the construction of pMON6457 which encodes (15-125) hIL-3; it contains the araBAD promoter and the lamB signal peptide fused to the variant hIL-3 amino acids 15-125.

FIG. 13 shows the construction of pMON6458; it contains the araBAD promoter and the lamb signal peptide fused to the variant hIL-3 amino acids 15-125.

FIG. 14 shows the construction of pMON6467 in which the bases encoding amino acids 35-40 of hIL-3 were deleted using site-directed PCR mutagenesis methods. pMON6467 was used as the template for the generation of single amino acid variants at positions 35-40 of hIL-3.

FIG. 15 shows the construction of single amino acid substitutions at position 35 of hIL-3 using site-directed PCR mutagenesis methods. The mutagenesis results in 20 different single amino substitutions, which is referred to as a “library”, at position 35 of hIL-3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to muteins of human interleukin-3 (hIL-3) in which amino acid substitutions have been made at from one to three positions in the amino acid sequence of the polypeptide and to hIL-3 muteins which have substantially the same structure and substantially the same biological activity. Preferred muteins of the present invention are (15-125) hIL-3 deletion mutants which have deletions of amino acids 1 to 14 at the N-terminus and/or 126 to 133 at the C-terminus and which both also have from one to three amino acid substitutions in the polypeptide and muteins having substantially the same structure and substantially the same biological activity. As used herein human interleukin-3 corresponds to the amino acid sequence (1-133) as depicted in FIG. 1 and (15-125) hIL-3 Corresponds to the 15 to 125 amino acid sequence of the hIL-3 polypeptide. Naturally occurring variants of hIL-3 polypeptide amino acids are also included in the present invention (for example, the allele in which proline rather than serine is at position 8 in the hIL-3 polypeptide sequence) as are variant hIL-3 molecules which are modified post-translationally (e.g. glycosylation).

The present invention also includes the DNA sequences which code for the mutant polypeptides, DNA sequences which are substantially similar and perform substantially the same function, and DNA sequences which differ from the DNAs encoding the muteins of the invention only due to the degeneracy of the genetic code.

Included in the present invention are novel mutant human interleukin-3 polypeptides comprising a polypeptide having the amino acid sequence of native human interleukin-3 wherein amino acids 126 to 133 have been deleted from the C-terminus of the native human interleukin-3 polypeptide and amino acids 1 to 14 have been deleted from the N-terminus of the native human interleukin-3 polypeptide and, in addition, polypeptides of the present invention also have one to three amino acid substitutions in the polypeptide sequence. The muteins of the present invention can have from one to three amino acid substitutions in the hIL-3 polypeptide chain and, in addition, can have deletions of amino acids at the N-terminus and/or the C-terminus.

Also included in the present invention are the DNA sequences coding for the muteins of the present invention; the oligonucleotide intermediates used to construct the mutant DNAs; and the polypeptides coded for by these oligonucleotides. These polypeptides may be useful as antagonists or as antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols.

The mutant hIL-3 polypeptides of the present invention may also have methionine, alanine, or methionine-alanine residues inserted at the N-terminus.

The present invention includes hIL-3 mutant polypeptides of the formula I:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn [SEQ ID NO:15]  1               5                   10                  15 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  20                  25                  30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa                  35                  40                  45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  50                  55                  60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  65                  70                  75 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  80                  85                  90 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  95                 100                 105 Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 110                 115                 120 Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe                 125                 130

wherein

Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg:

Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;

Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;

Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;

Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;

Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;

Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;

Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;

Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;

Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;

Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;

Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;

Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu:

Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;

Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;

Xaa at position 36 is Asp, Leu, or Val;

Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 38 is Asn, or Ala;

Xaa at position 40 is Leu, Trp, or Arg;

Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;

Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;

Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;

Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;

Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;

Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;

Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;

Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;

Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;

Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;

Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;

Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;

Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His , Ala or Leu;

Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;

Xaa at position 57 is Asn or Gly;

Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;

Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg;

Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;

Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;

Xaa at position 62 is Asn His, Val, Arg, Pro, Thr, Asp, or Ile;

Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;

Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;

Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;

Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;

Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;

Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;

Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;

Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;

Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;

Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;

Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu:

Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;

Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;

Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;

Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 80 is Agn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;

Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;

Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;

Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;

Xaa at position 85 is Leu, Asn, Val, or Gln;

Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;

Xaa at position 87 is Leu, Ser, Trp, or Gly;

Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;

Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;

Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;

Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;

Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;

Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;

Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro;

Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr;

Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;

Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;

Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;

Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;

Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;

Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln;

Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;

Xaa at position 103 is Asp, or Ser;

Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;

Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;

Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;

Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;

Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp;

Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;

Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;

Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;

Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;

Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;

Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;

Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;

Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;

Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;

Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 22 is Leu, and/or Xaa at position 34 is Gly or Glu, and/or Xaa at position 44 is Ala, and/or Xda at position 46 is Lys or Ala, and/or Xaa at position 50 is Lys, and/or Xaa at position 59 is Pro or Arg, and/or Xaa at position 63 is Lys, and/or Xaa at position 75 is Gly or Arg, and/or Xaa at position 94 is Pro, and/or Xaa at position 98 is Arg, and/or Xaa at position 106 is Lys, and/or Xaa at position 110 is Ala or Glu, and/or Xaa at position 111 is Met, then there must be at least one additional substitution besides the ones indicated.

Included in the present invention are (1-133) hIL-3 mutant polypeptides of the Formula II:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn (SEQ ID NO:16)  1               5                   10                 15 Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa                 20                   25                 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa                 35                   40                 45 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa                 50                   55                 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 65                   70                 75 Xaa Xaa Leu Xaa kaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa                 80                   85                 90 Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa                 95                  100                105 Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa Xaa                 110                 115                120 Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe                 125                 130

wherein

Xaa at position 17 is Ser, Gly, Asp, Met, or Gln; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 19 is Met, Phe, Ile, Arg, or Ala;

Xaa at position 20 is Ile or Pro;

Xaa at position 21 is Asp or Glu;

Xaa at position 21 is Ile, Val, Ala, Leu, or Gly;

Xaa at position 24 is Ile, Val, Phe, or Leu;

Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 26 is His, Phe, Gly, Arg, or Ala;

Xaa at position 28 is Lys, Leu, Gln, Gly, Pro, or Val;

Xaa at position 29 is Gln, Asn, Leu, Arg, or Val;

Xaa at position 30 is Pro, His, Thr, Gly, or Gln;

Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 33 is Pro, Leu, Gln, Ala, or Glu;

Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 36 is Asp or Leu;

Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 38 is Asn or Ala;

Xaa at position 41 is Asn, Cys, Arg, His, Met, or Pro;

Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, Val or Arg;

Xaa at position 44 is Asp or Glu;

Xaa at position 45 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn, Glu, Ser, or Trp;

Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Gly;

Xaa at position 47 is Ile, Val, or His;

Xaa at position 49 is Met, Asn, or Asp;

Xaa at position 50 is Glu, Thr, Ala, Asn, Ser or Asp;

Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 52 is Asn or Gly;

Xaa at position 53 is Leu, Met, or Phe;

Xaa at position 54 is Arg, Ala, or Ser;

Xaa at position 55 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val or Lys;

Xaa at position 59 is Glu, Tyr, His, Leu, or Arg;

Xaa at position 60 is Ala, Ser, Asn, or Thr;

Xaa at position 61 is Phe or Ser;

Xaa at position 62 is Asn, Val, Pro, Thr, or Ile;

Xaa at position 63 is Arg, Tyr, Lys, Ser, His, or Val;

Xaa at position 64 is Ala or Asn;

Xaa at position 65 is Val, Thr, Leu, or Ser;

Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 67 is Ser, Phe, Val, Gly, Asn, Ile, or His:

Xaa at position 68 is Leu, Val, Ile, Phe, or His;

Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 70 is Asn or Pro;

Xaa at position 71 is Ala, Met, Pro, Arg, Glu, Thr, or Gln;

Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro;

Xaa at position 74 is Ile or Met;

Xaa at position 75 is Glu, Gly, Asp, Ser, or Gln;

Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp;

Xaa at position 77 is Ile, Ser, or Leu;

Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 80 is Asn, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 81 is Leu, or Val;

Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 83 is Pro, Ala, Thr, Trp, or Met;

Xaa at position 85 is Leu or Val;

Xaa at position 87 is Leu or Ser;

Xaa at position 88 is Ala, Arg, or Trp;

Xaa at position 89 is Thr, Asp, Glu, His, Asn, or Ser;

Xaa at position 90 is Ala, Asp, or Met;

Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp;

Xaa at position 92 is Pro or Ser;

Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser or Thr;

Xaa at position 96 is Pro or Tyr;

Xaa at position 97 is Ile, Val, or Ala;

Xaa at position 98 is His, Ile, Asn, Asp, Ala, Thr, Leu, Arg, Gln, Glu, lys, Met, Ser, Tyr, Val or Pro;

Xaa at position 99 is Ile, Leu, Val, or Phe;

Xaa at position 100 is Lys, Leu, His, Arg, Ile, Gln, Pro, or Ser;

Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu or Tyr;

Xaa at position 102 is Gly, Glu, Lys, or Ser;

Xaa at position 104 is Trp, Val, Tyr, Met, or Leu;

Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 106 is Glu, Ser, Ala, or Gly;

Xaa at position 108 is Arg, Ala, Gln, Ser or Lys;

Xaa at position 109 is Arg, Thr, Glu, Leu, Ser, or Gly;

Xaa at position 112 is Thr, Val, Gln, Glu, His, or Ser;

Xaa at position 114 is Tyr or Trp;

Xaa at position 115 is Leu or Ala;

Xaa at position 116 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr or Ile;

Xaa at position 117 is Thr, Ser, or Asn;

Xaa at position 119 is Glu, Ser, Pro, Leu, Thr, or Tyr;

Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 34 is Gly or/and Xaa or position 46 is Lys or Ala or/and Xaa at position 59 is Arg and/or Xaa at position 63 is Lys and/or Xaa at position 75 is Gly and/or Xaa at position 98 is Arg then there must be at least one additional substitution besides the ones indicated.

Included in the present invention are (1-133) hIL-3 mutant polypeptides of the Formula III:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn (SEQ ID NO:17)  1               5                   10                 15 Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa                  20                  25                 30 Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa                  35                  40                 45 Xaa Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa                  50                  55                 60 Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu                  65                  70                 75 Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala                  80                  85                 90 Xaa Pro Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa                  95                 100                105 Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu Xaa                 110                 115                120 Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe                 125                 130

wherein

Xaa at position 17 is Ser, Gly, Asp, Met, or Gln;

Xaa at position 18 is Asn, His, or Ile;

Xaa at position 19 is Met or Ile;

Xaa at position 21 is Asp or Glu;

Xaa at position 23 is Ile, Ala, Leu, or Gly;

Xaa at position 24 is Ile, Val, or Leu;

Xaa at position 25 is Thr, His, Gln, or Ala;

Xaa at position 26 is His or Ala;

Xaa at position 29 is Gln, Asn, or Val;

Xaa at position 30 is Pro, Gly, or Gln;

Xaa at position 31 is Pro, Asp, Gly, or Gln;

Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 33 is Pro or Glu;

Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 37 is Phe, Ser, Pro, or Trp;

Xaa at position 38 is Asn or Ala;

Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

Xaa at position 44 is Asp or Glu;

Xaa at position 45 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or Lys;

Xaa at position 46 is Asp, Phe, Ser, Thr, Ala, Asn Gln, Glu, His, Ile, Lys, Tyr, Val or Cys;

Xaa at position 50 is Glu, Ala, Asn, Ser or Asp;

Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 54 is Arg or Ala;

Xaa at position 54 is Arg or Ala;

Xaa at position 55 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val or Lys;

Xaa at position 60 is Ala or Ser;

Xaa at position 62 is Asn, Pro, Thr, or Ile;

Xaa at position 63 is Arg or Lys;

Xaa at position 64 is Ala or Asn;

Xaa at position 65 is Val or Thr;

Xaa at position 66 is Lys or Arg;

Xaa at position 67 is Ser, Phe, or His;

Xaa at position 68 is Leu, Ile, Phe, or His;

Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 71 is Ala, Pro, or Arg;

Xaa at position 72 is Ser, Glu, Arg, oz Asp;

Xaa at position 73 is Ala or Leu;

Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;

Xaa at position 77 is Ile or Leu;

Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 80 is Asn, Gly, Glu, or Arg;

Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 83 is Pro or Thr;

Xaa at position 85 is Leu or Val;

Xaa at position 87 is Leu or Ser;

Xaa at position 88 is Ala or Trp;

Xaa at position 91 is Ala or Pro;

Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser or Thr;

Xaa at position 96 is Pro or Tyr;

Xaa at position 97 is Ile or Val;

Xaa at position 98 is His, Ile, Asn, Leu, Ala, Thr, Arg, Gln, Lys, Met, Ser, Tyr, Val or Pro;

Xaa at position 99 is Ile, Leu, or Val;

Xaa at position 100 is Lys, Arg, Ile, Gln, Pro, or Ser;

Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Pro, Asn, Ile, Leu or Tyr;

Xaa at position 104 is Trp or Leu;

Xaa at position 105 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 106 is Glu or Gly;

Xaa at position 108 is Arg, Ala, or Ser;

Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;

Xaa at position 112 is Thr, Val, or Gln;

Xaa at position 114 is Tyr or Trp;

Xaa at position 115 is Leu or Ala;

Xaa at position 116 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr or Ile;

Xaa at position 117 is Thr or Ser;

Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;

Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 22 is Leu, and/or Xaa at position 34 is Gly or Glu, and/or Xaa at position 44 is Ala, and/or Xaa at position 46 is Lys or Ala, and/or Xaa at position 50 is Lys, and/or Xaa at position 59 is Pro or Arg, and/or Xaa at position 63 is Lys, and/or Xaa at position 75 is Gly or Arg, and/or Xaa at position 94 is Pro, and/or Xaa at position 98 is Arg, and/or Xaa at position 106 is Lys, and/or Xaa at position 110 is Ala or Glu, and/or Xaa at position 111 is Met, then there must be at least one additional substitution besides the ones indicated. and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 34 is Gly and/or Xaa at position 46 is Lys or Ala, and/or Xaa at position 63 is Lys, and/or Xaa at position 98 is Arg, then two or three of the amino acid designated by Xaa are different from the corresponding amino acids of the native (1-133) human interleukin-3.

Included in the present invention are (1-133) hIL-3 mutant polypeptides of the Formula IV:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn [SEQ ID NO:18]  1               5                   10                 15 Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa                  20                  25                 30 Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa                  35                  40                 45 Xaa Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala                  50                  55                 60 Phe Xaa Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu                  65                  70                 75 Xaa Xaa Leu Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala                  80                  85                 90 Xaa Pro Xaa Arg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa                  95                 100                105 Glu Phe Xaa Xaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa                 110                 115                120 Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe                 125                 130

wherein

Xaa at position 17 is Ser, Gly, Asp, or Gln;

Xaa at position 18 is Asn, His, or Ile;

Xaa at position 23 is Ile, Ala, Leu, or Gly;

Xaa at position 25 is Thr, His, or Gln;

Xaa at position 26 is His or Ala;

Xaa at position 29 is Gln or Asn;

Xaa at position 30 is Pro or Gly;

Xaa at position 32 is Leu, Arg, Asn, or Ala;

Xaa at position 34 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met;

Xaa at position 35 is Leu, Ala, Asn, or Pro;

Xaa at position 29 is Asn or Ala;

Xaa at position 42 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

Xaa at position 45 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys;

Xaa at position 46 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;

Xaa at position 50 is Glu Asn, Ser or Asp;

Xaa at position 51 is Asn, Arg, Pro, Thr, or His;

Xaa at position 55 is Arg, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;

Xaa at position 62 is Asn, Pro, or Thr;

Xaa at position 64 is Ala or Asn;

Xaa at position 65 is Val or Thr;

Xaa at position 67 is Ser or Phe;

Xaa at position 68 is Leu or Phe;

Xaa at position 69 is Gln, Ala, Glu, or Arg;

Xaa at position 76 is Ser, Val, Asn, Pro, or Gly;

Xaa at position 77 is Ile or Leu;

Xaa at position 79 is Lys, Asn, Met, Arg, Ile, or Gly;

Xaa at position 80 is Asn, Gly, Glu, or Arg;

Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 87 is Leu or Ser;

Xaa at position 88 is Ala or Trp;

Xaa at position 91 is Ala or Pro;

Xaa at position 93 is Thr, Asp, or Ala;

Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;

Xaa at position 98 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;

Xaa at position 99 is Ile or Leu;

Xaa at position 100 is Lys or Arg;

Xaa at position 101 is Asp, Pro, Met, Lys, Thr, His, Pro, Asn, Ile, Leu or Tyr;

Xaa at position 105 is Asn, Pro, Ser, Ile or Asp;

Xaa at position 108 is Arg, Ala, or Ser;

Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;

Xaa at position 112 is Thr or Gln;

Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, Tyr or Ile;

Xaa at position 117 is Thr or Ser;

Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Pro, or Asp;

Xaa at position 122 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;

Xaa at position 123 is Ala, Met, Glu, Ser, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3.

Preferred polypeptides of the present invention are (15-125) hIL-3 mutant polypeptides of the Formula V:

Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa [SEQ ID NO:19] 1                5                   10                 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa                  20                  25                 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  35                  40                 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  50                  55                 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  65                  70                 75 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  80                  85                 90 Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  95                 100                105 Xaa Xaa Xaa Xaa Gln Gln                     110 wherein

wherein

Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;

Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;

Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;

Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;

Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly;

Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;

Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;

Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp;

Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala;

Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;

Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val;

Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;

Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu;

Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;

Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;

Xaa at position 22 is Asp, Leu, or Val;

Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 24 is Asn, or Ala;

Xaa at position 26 is Leu, Trp, or Arg;

Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro;

Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met;

Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;

Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr,Met, Trp, Glu, Asn, Gln, Ala or Pro;

Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp;

Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;

Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His;

Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;

Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;

Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;

Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr;

Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, Met, or;

Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;

Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;

Xaa at position 43 is Asn or Gly;

Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;

Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg;

Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr;

Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;

Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;

Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;

Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys;

Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser;

Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;

Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;

Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;

Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala;

Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;

Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;

Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala;

Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;

Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;

Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu;

Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;

Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp;

Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;

Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;

Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met;

Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val;

Xaa at position 71 is Leu, Asn, Val, or Gln;

Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys;

Xaa at position 73 is Leu, Ser, Trp, or Gly;

Xaa at position 74 is Ala, Lys, Arg, Val, or Trp;

Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;

Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;

Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;

Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;

Xaa at position 79 iS Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;

Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro;

Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr;

Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr:

Xaa at position 83 is Ile, Val, Lys, Ala, or Asn;

Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;

Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;

Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, Pro;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu or Gln;

Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;

Xaa at position 89 is Asp, or Ser;

Xaa at position 90 Is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;

Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;

Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro;

Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;

Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp;

Xaa at position 97 is Leu, Ile, Arg, Asp, or Met;

Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;

Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lsy, Leu, Ile, Val or Asn;

Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;

Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;

Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;

Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;

Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;

Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;

Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Included in the present invention are (15-125) hIL-3 mutant polypeptides of the Formula VI:

Asn Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa [SEQ ID NO:20]  1               5                   10                 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa                  20                  25                 30 Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa                  35                  40                 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                  50                  55                 60 Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa                  65                  70                 75 Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa                  80                  85                 90 Xaa Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa                  95                 100                105 Xaa Xaa Xaa Xaa Gln Gln                 110

wherein

Xaa at position 3 is Ser, Gly, Asp, Met, or Gln;

Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 5 is Met, Phe, Ile, Arg, or Ala;

Xaa at position 6 is Ile or Pro;

Xaa at position 7 is Asp, or Glu;

Xaa at position 9 is Ile, Val, Ala, Leu, or Gly;

Xaa at position 10 is Ile, Val, Phe, or Leu;

Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 12 is His, Phe, Gly, Arg, or Ala;

Xaa at position 14 is Lys, Leu, Gln, Gly, Pro, or Val;

Xaa at position 15 is Gln, Asn, Leu, Arg, or Val;

Xaa at position 16 is Pro, His, Thr, Gly, or Gln;

Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 19 is Pro, Leu, Gln, Ala, or Glu;

Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 22 is Asp or Leu;

Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 24 is Asn or Ala;

Xaa at position 27 is Asn, Cys, Arg, His, Met, or Pro;

Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg;

Xaa at position 30 is Asp, or Glu:

Xaa at position 31 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn Glu, Ser or Trp;

Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Gly;

Xaa at position 33 is Ile, Val, or His;

Xaa at position 35 is Met, Asn, or Asp;

Xaa at position 36 is Glu, Thr, Ala, Asn, Ser or Asp;

Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 38 is Asn or Gly;

Xaa at position 39 is Leu, Met, or Phe;

Xaa at position 40 is Arg, Ala or Ser;

Xaa at position 41 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val or Lys;

Xaa at position 45 is Glu, Tyr, His, Leu, or Arg;

Xaa at position 46 is Ala, Ser, Asn, or Thr;

Xaa at position 47 is Phe or Ser;

Xaa at position 48 is Asn, Val, Pro, Thr, or Ile;

Xaa at position 49 is Arg, Tyr, Lys, Ser, His, or Val;

Xaa at position 50 is Ala or Asn;

Xaa at position 51 is Val, Thr, Leu, or Ser;

Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 53 is Ser, Phe, Val, Gly, Asn, Ile, or His;

Xaa at position 54 is Leu, Val, Ile, Phe, or His;

Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 56 is Asn or Pro;

Xaa at position 57 is Ala, Met, Pro, Arg, Glu, Thr, or Gln;

Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro;

Xaa at position 60 is Ile or Met;

Xaa at position 61 is Glu, Gly, Asp, Ser, or Gln;

Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp;

Xaa at position 63 is Ile, Ser, or Leu;

Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp;

Xaa at position 66 is Asn, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 67 is Leu, or Val;

Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 69 is Pro, Ala, Thr, Trp, or Met;

Xaa at position 71 is Leu or Val;

Xaa at position 73 is Leu or Ser;

Xaa at position 74 is Ala, Arg, or Trp:

Xaa at position 75 is Thr, Asp, Glu, His, Asn, or Ser;

Xaa at position 76 is Ala, Asp, or Met;

Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp;

Xaa at position 78 is Pro or Ser;

Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser or Thr;

Xaa at position 82 is Pro or Tyr;

Xaa at position 83 is Ile, Val, or Ala;

Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Arg, Gln, Glu, Lys, Met, per, Tyr, Val or Pro;

Xaa at position 85 is Ile, Leu, Val, or Phe;

Xaa at position 86 is Lys, Leu, His, Arg, Ile, Gln, Pro or Ser;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu or Tyr;

Xaa at position 99 is Gly, Glu, Lys, or Ser;

Xaa at position 90 is Trp, Val, Tyr, Met, or Leu;

Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 92 is Glu, Ser, Ala, or Gly;

Xaa at position 94 is Arg, Ala, Gln, Ser or Lys;

Xaa at position 95 is Arg, Thr, Glu, Leu, Ser, or Gly;

Xaa at position 98 is Thr, Val, Gln, Glu, His, or Ser;

Xaa at position 100 is Tyr or Trp;

Xaa at position 101 is Leu or Ala;

Xaa at position 102 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr or Ile;

Xaa at position 103 is Thr, Ser, or Asn;

Xaa at position 105 is Glu, Ser, Pro, Leu, Thr, or Tyr;

Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Included in the present invention are (15-125) hIL-3 mutant polypeptides of the Formula VII:

Asn Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa [SEQ ID NO:21]  1               5                   10                 15 Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa                  20                  25                 30 Xaa Xaa Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu                  35                  40                 45 Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile                  50                  55                 60 Glu Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr                  65                  70                 75 Ala Xaa Pro Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa                  80                  85                 90 Xaa Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu                  95                 100                105 Xaa Xaa Xaa Xaa Gln Gln                 110

wherein

Xaa at position 3 is Ser, Gly, Asp, Met, or Gln;

Xaa at position 4 is Asn, His, or Ile;

Xaa at position 5 is Met or Ile;

Xaa at position 7 is Asp or Glu;

Xaa at position 9 is Ile, Ala, Leu, or Gly;

Xaa at position 10 is Ile, Val, or Leu;

Xaa at position 11 is Thr, His, Gln, or Ala;

Xaa at position 12 is His or Ala;

Xaa at position 15 is Gln, Asn, or Val;

Xaa at position 16 is Pro, Gly, or Gln;

Xaa at position 17 is Pro, Asp, Gly, or Gln;

Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 19 is Pro or Glu;

Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 23 is Phe, Ser, Pro, or Trp;

Xaa at position 24 is Asn or Ala;

Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met Tyr or Arg;

Xaa at position 30 is Asp or Glu;

Xaa at position 31 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or Lys;

Xaa at position 32 is Asp, Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Cys;

Xaa at position 36 is Glu, Ala, Asn, Ser or Asp;

Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 40 is Arg or Ala;

Xaa at position 41 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val or Lys;

Xaa at position 46 is Ala or Ser;

Xaa at position 48 is Asn, Pro, Thr, or ale;

Xaa at position 49 is Arg or Lys;

Xaa at position 50 is Ala or Asn;

Xaa at position 51 is Val or Thr;

Xaa at position 52 is Lys or Arg;

Xaa at position 53 is Ser, Phe, or His;

Xaa at position 54 is Leu, Ile, Phe, or His;

Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 57 is Ala, Pro, or Arg;

Xaa at position 58 is Ser, Glu, Arg, or Asp;

Xaa at position 59 is Ala or Leu;

Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;

Xaa at position 63 is Ile or Leu;

Xaa at position 65 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 66 is Asn, Gly, Glu, or Arg;

Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 69 is Pro or Thr;

Xaa at position 71 is Leu or Val;

Xaa at position 73 is Leu or Ser;

Xaa at position 74 is Ala or Trp;

Xaa at position 77 is Ala or Pro;

Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser or Thr;

Xaa at position 82 is Pro or Tyr;

Xaa at position 83 is Ile or Val;

Xaa at position 84 is His, Ile, Asn, Ala, Thr, Leu, Arg, Gln, Lys, Met, Ser, Tyr, Val or Pro;

Xaa at position 85 is Ile, Leu, or Val;

Xaa at position 86 is Lys, Arg, Ile, Gln, Pro, or Ser;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Asn, Ile, Leu or Tyr;

Xaa at position 90 is Trp or Leu;

Xaa at position 91 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 92 is Glu, or Gly;

Xaa at position 94 is Arg, Ala, or Ser;

Xaa at position 95 iS Arg, Thr, Glu, Leu, or Ser;

Xaa at position 98 is Thr, Val, or Gln;

Xaa at position 100 is Tyr or Trp;

Xaa at position 101 is Leu or Ala;

Xaa at position 102 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr or Ile;

Xaa at position 103 is Thr or Ser;

Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;

Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Included in the present invention are (15-125) hIL-3 mutant polypeptides of the Formula VIII:

Asn Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa [SEQ ID NO:22]  1               5                   10                 15 Xaa Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp                  20                  25                 30 Xaa Xaa Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu                  35                  40                 45 Ala Phe Xaa Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile                  50                  55                 60 Glu Xaa Xaa Leu Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr                  65                  70                 75 Ala Xaa Pro Xaa Arg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp                  80                  85                 90 Xaa Glu Phe Xaa Xaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu                  95                 100                 105 Xaa Xaa Xaa Xaa Gln Gln                 110

wherein

Xaa at position 3 is Ser, Gly, Asp, or Gln;

Xaa at position 4 is Asn, His, or Ile;

Xaa at position 9 is Ile, Ala, Leu, or Gly;

Xaa at position 11 is Thr, His, or Gln;

Xaa at position 12 is His or Ala;

Xaa at position 15 is Gln or Asn;

Xaa at position 16 is Pro or Gly;

Xaa at position 18 is Leu, Arg, Asn, or Ala;

Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 21 is Leu, Ala, Asn, or Pro;

Xaa at position 24 is Asn or Ala:

Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;

Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;

Xaa at position 36 is Glu, Asn, Ser or Asp;

Xaa at position 37 is Asn, Arg, Pro, Thr, or His;

Xaa at position 41 is Arg, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;

Xaa at position 48 is Asn, Pro, or Thr;

Xaa at position 50 is Ala or Asn;

Xaa at position 51 is Val or Thr;

Xaa at position 53 is Ser or Phe;

Xaa at position 54 is Leu or Phe;

Xaa at position 55 is Gln, Ala, Glu, or Arg;

Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;

Xaa at position 63 is Ile or Leu;

Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;

Xaa at position 66 is Asn, Gly, Glu, or Arg;

Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 73 is Leu or Ser;

Xaa at position 74 is Ala or Trp;

Xaa at position 77 is Ala or Pro;

Xaa at position 79 is Thr, Asp, or Ala;

Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;

Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;

Xaa at position 85 is Ile or Leu;

Xaa at position 86 is Lys or Arg;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Pro, Asn, Ile, Leu or Tyr;

Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;

Xaa at position 94 is Arg, Ala, or Ser;

Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;

Xaa at position 98 is Thr or Gln;

Xaa at position 102 is Lys, Val, Trp, or Ile;

Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;

Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;

Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;

Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;

and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133)human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

In Formulas V, VI, VII and VIII the Asn in position 1 corresponds to the Asn in position 15 of native hIL-3 and positions 1 to 111 correspond to positions 15 to 125 in the native hIL-3 sequence shown in FIG. 1.

Also included in the present invention are polypeptides of the following formula (IX):

        1               5                  10 [SEQ ID NO:129] (Met)_(m)-Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr             15                   20 Ser Trp Val Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile 25                  30                   35 Xaa His Leu Lys Xaa Pro Pro Xaa Pro Leu Leu Asp Xaa         40                  45                   50 Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu Met Glu                 55                   60 Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg     65                  70                  75 Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa             80                   85 Ile Leu Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr 90                   95                 100 Ala Ala Pro Xaa Arg His Pro Ile Xaa Ile Lys Xaa Gly         105                 110                 115 Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr Phe Tyr Leu                 120                 125 Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln Thr Thr Leu     130 Ser Leu Ala Ile Phe

wherein m is 0 or 1; Xaa at position 18 is Asn or Ile; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, or Val; Xaa at position 32 is Leu, Ala, or Asn; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 42 is Glu, Ala, or Ser; Xaa at position 45 is Gln, Val, or Met; Xaa at position 51 is Asn or Arg; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn or Val; Xaa at position 67 is Ser, Asn, or His; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser or Ala; Xaa at position 79 is Lys or Arg; Xaa at position 82 is Leu, Glu, or Val; Xaa at position 87 is Leu or Ser; Xaa at position 93 is Pro or Ser; Xaa at position 98 is His, Ile, or Thr; Xaa at position 101 is Asp or Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg or Glu; Xaa at position 116 is Lys or Val; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Polypeptides of the present invention include those (15-125) hIL-3 muteins of the following formula (X):

              1                5                  10 [SEQ ID NO:130] (Met_(m)-Ala_(n))p-Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile                 15                   20 Xaa His Leu Lys Xaa Pro Pro Xaa Pro Leu Leu Asp Xaa     25                   30                   35 Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu Met Glu             40                   45 Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg  50                 55                   60 Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa         65                   70                  75 Ile Leu Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr                 80                   85 Ala Ala Pro Xaa Arg His Pro Ile Xaa Ile Lys Xaa Gly      90                  95                  100 Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr Phe Tyr Leu             105                  110 Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln

wherein m is 0 or 1; n is 0 or 1; p is 0 or 1; Xaa at position 4 is Asn or Ile; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, or Val; Xaa at position 18 is Leu, Ala, or Asn; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 28 is Glu, Ala, or Ser; Xaa at position 31 is Gln, Val, or Met; Xaa at position 37 is Asn or Arg; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn or Val; Xaa at position 53 is Ser, Asn, or His; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser or Ala; Xaa at position 65 is Lys or Arg; Xaa at position 68 is Leu, Glu, or Val; Xaa at position 73 is Leu or Ser; Xaa at position 79 is Pro or Ser; Xaa at position 84 is His, Ile, or Thr; Xaa at position 87 is Asp or Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg or Glu; Xaa at position 102 is Lys or Val,; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu;

wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125)human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

The present invention includes polypeptides of Formula IX and Formula X above wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3 or native (15-125) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

“Mutant amino acid sequence,” “mutant protein” or “mutant polypeptide” refers to a polypeptide having an amino acid sequence which varies from a native sequence or is encoded by a nucleotide sequence intentionally made variant from a native sequence. “Mutant protein,” “variant protein” or “mutein” means a protein comprising a mutant amino acid sequence and includes polypeptides which differ from the amino acid sequence of native hIL-3 due to amino acid deletions, substitutions, or both. “Native sequence” refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.

Human IL-3 can be characterized by its ability to stimulate colony formation by human hematopoietic progenitor cells. The colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic macrophages and mixtures thereof. Human IL-3 has demonstrated an ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans (Gillio, A. P., et al. (1990); Ganser, A., et al. (1990); Falk, S., et al. (1991). Additional activities of hIL-3 include the ability to stimulate leukocyte migration and chemotaxis; the ability to prime human leukocytes to produce high levels of inflammatory mediators like leukotrienes and histamine; the ability to induce cell surface expression of molecules needed for leukocyte adhesion; and the ability to trigger dermal inflammatory responses and fever. Many or all of these biological activities of hIL-3 involve signal transduction and high affinity receptor binding. Mutant polypeptides of the present invention may exhibit useful properties such as having similar or greater biological activity when compared to native hIL-3 or by having improved half-life or decreased adverse side effects, or a combination of these properties. They may also be useful as antagonists. hIL-3 mutant polypeptides which have little or no activity when compared to native hIL-3 may still be useful as antagonists, as antigens for the production of antibodies for use in immunology or immunotherapy, as genetic probes or as intermediates used to construct other useful hIL-3 muteins. Since hIL-3 functions by binding to its receptor(s) and triggering second messages resulting in competent signal transduction, hIL-3 muteins of this invention may be useful in helping to determine which specific amino acid sequences are responsible for these activities.

The novel hIL-3 mutant polypeptides of the present invention will preferably have at least one biological property of human IL-3 or of an IL-3-like growth factor and may have more than one IL-3-like biological property, or an improved property, or a reduction in an undesirable biological property of human IL-3. Some mutant polypeptides of the present invention may also exhibit an improved side effect profile. For example, they may exhibit a decrease in leukotriene release or histamine release when compared to native hIL-3 or (15-125) hIL-3. Such hIL-3 or hIL-3-like biological properties may include one or more of the following biological characteristics and in vivo and in vitro activities.

One such property is the support of the growth and differentiation of progenitor cells committed to erythroid, lymphoid, and myeloid lineages. For example, in a standard human bone marrow assay, an IL-3-like biological property is the stimulation of granulocytic type colonies, megakaryocytic type colonies, monocyte/macrophage type colonies, and erythroid bursts. Other IL-3-like properties are the interaction with early multipotential stem cells, the sustaining of the growth of pluripotent precursor cells, the ability to stimulate chronic myelogenous leukemia (CML) cell proliferation, the stimulation of proliferation of mast cells, the ability to support the growth of various factor-dependent cell lines, and the ability to trigger immature bone marrow cell progenitors. Other biological properties of IL-3 have been disclosed in the art. Human IL-3 also has some biological activities which may in. some cases be undesirable, for example the ability to stimulate leukotriene release and the ability to stimulate increased histamine synthesis in spleen and bone marrow cultures and in vivo.

Biological activity of hIL-3 and hIL-3 mutant proteins of the present invention is determined by DNA synthesis by human acute myelogenous leukemia cells (AML). The factor-dependent cell line AML 193 was adapted for use in testing biological activity.

One object of the present invention is to provide hIL-3 muteins and hIL-3 deletion muteins with one or more amino acid substitutions in the polypeptide sequence which have similar of improved biological activity in relation to native hIL-3 or native (15-125) hIL-3.

The present invention includes mutant polypeptides comprising minimally amino acid residues 15 to 118 of hIL-3 with or without additional amino acid extensions to the N-terminus and/or C-terminus which further contain from one to three or more amino acid substitutions in the amino acid sequence of the polypeptide. It has been found that the (15-125) hIL-3 mutant is more soluble than is hIL-3 when expressed in the cytoplasm of E. coli, and the protein is secreted to the periplasm in E. coli at higher levels compared to native hIL-3.

When expressed in the E. coli cytoplasm, the above-mentioned mutant hIL-3 polypeptides of the present invention may also be constructed with Met-Ala- at the N-terminus so that upon expression the Met is cleaved off leaving Ala at the N-terminus. These mutant hIL-3 polypeptides may also be expressed in E. coli by fusing a signal peptide to the N-terminus. This signal peptide is cleaved from the polypeptide as part of the secretion process. Secretion in E. coli can be used to obtain the correct amino acid at the N-terminus (e.g., Asn¹⁵ in the (15-125) hIL-3 polypeptide) due to the precise nature of the signal peptidase. This is in contrast to the heterogeneity often observed at the N-terminus of proteins expressed in the cytoplasm in E. coli.

The hIL-3 mutant polypeptides of the present invention may have hIL-3 or hIL-3-like activity. For example, they may possess one or more of the biological activities of native hIL-3 and may be useful in stimulating the production of hematopoietic cells by human or primate progenitor cells. The hIL-3 muteins of the present invention and pharmaceutical compositions containing them may be useful in the treatment of conditions in which hematopoietic cell populations have been reduced or destroyed due to disease or to treatments such as radiation or chemotherapy.

hIL-3 muteins of the present invention may also be useful as antagonists which block the hIL-3 receptor by binding specifically to it and preventing binding of the agonist.

One potential advantage of the (15-125) hIL-3 muteins of the present invention, particularly those which retain activity similar to or better than that of native hIL-3, is that it may be possible to use a smaller amount of the biologically active mutein to produce the desired therapeutic effect. This may make it possible to reduce the number of treatments necessary to produce the desired therapeutic effect. The use of smaller amounts may also reduce the possibility of any potential antigenic effects or other possible undesirable side effects. For example, if a desired therapeutic effect can be achieved with a smaller amount of polypeptide it may be possible to reduce or eliminate side effects associated with the administration of native IL-3 such as the stimulation of leukotriene and/or histamine release. The hIL-3 muteins of the present invention may also be useful in the activation of stem cells or progenitors which have low receptor numbers. Pharmaceutical compositions containing hIL-3 muteins of the present invention can be administered parenterally, intravenously, or subcutaneously.

In variants which contain an additional cysteine the presence of the cysteine permits the labeling of the protein with ricin which permits targeting ricin and other toxins or tracers using a sulfhydryl linkage to the hIL-3 receptor.

As another aspect of the present invention, there is provided a novel method for producing the novel family of human IL-3 muteins. The method of the present invention involves culturing a suitable cell or cell line( which has been transformed with a vector containing a DNA sequence coding for expression of a novel hIL-3 mutant polypeptide. Suitable cells or cell lines may be bacterial cells. For example, the various strains of E. coli are well-known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM101 [Yanish-Perron, et al. (1985)] and MON105 [Obukowicz, et al. (1992)]. Various strains of B subtilis may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention.

Also suitable for use in the present invention are mammalian cells, such as Chinese hamster ovary cells (CHO). General methods for expression of foreign genes in mammalian cells are reviewed in; Kaufman, R. J. (1987) High level production of proteins-in mammalian cells, in Genetic Engineering, Principles and Methods, Vol. 9, J. K. Setlow, editor, Plenum Press, New York. An expression vector is constructed in which a strong promoter capable of functioning in mammalian cells drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally fused to the coding region for the hIL-3 variant. For example, plasmids such as pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, Calif.) can be used. The eukaryotic secretion signal peptide coding region can be from the hIL-3 gene itself or it can be from another secreted mammalian protein (Bayne, M. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84, 2638-2642). After construction of the vector containing the hIL-3 variant gene, the vector DNA is transfected into mammalian cells. Such cells can be, for example, the COS7, HeLa, BHK, CHO, or mouse L lines. The cells can be cultured, for example, in DMEM media (JRH Scientific). The hIL-3 variant secreted into the media can be recovered by standard biochemical approaches following transient expression 24-72 hours after transfection of the cells or after establishment of stable cell lines following selection for neomycin resistance. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 2:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cell line is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line.

Where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein. In addition, general methods for expression of-foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E. (1987)—A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555. An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally fused to the coding region for the hIL-3 variant polypeptide. For example, the plasmid pVL1392 (obtained from Invitrogen Corp., San Diego, Calif.) can be used. After construction of the vector carrying the hIL-3 variant gene, two micrograms of this DNA is cotransfected with one microgram of Baculovirus DNA (see Summers & Smith, 1987) into insect cells, strain SF9. Pure recombinant Baculovirus carrying the hIL-3 variant is used to infect cells cultured, for example, in Excell 401 serum-free medium (in Biosciences, Lenexa, Kans.). The hIL-3 variant secreted into the medium can be recovered by standard biochemical approaches.

Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel hIL-3 muteins. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform microorganisms capable of expressing the hIL-3 muteins include expression vectors comprising nucleotide sequences coding for the hIL-3 muteins joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.

Vectors incorporating modified sequences as described above are included in the present invention and are useful in the production of the hIL-3 mutant polypeptides. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.

The present invention also includes the construction and expression of (15-125)human interleukin-3 muteins having one or more amino acid substitutions in secretion vectors that optimize accumulation of correctly folded, active polypeptide. While many heterologous proteins have been secreted in E. coil there is still a great deal of unpredictability and limited success (Stader and Silhavy 1990). Full-length hIL-3 is such a protein, where attempts to secrete the protein in E. coli resulted in low levels of secretion. Secretion of the variant (15-125) hIL-3 mutant polypeptides of the present invention as a fusion with a signal peptide such as lamB results in correctly folded protein that can be removed from the periplasm of E. coli by osmotic shock fractionation. This property of the variant (15-125) hIL-3 muteins allows for the direct and rapid screening for bioactivity of the secreted material in the crude osmotic shock fraction, which is a significant advantage. Furthermore, it provides a means of using the (15-125) hIL-3 muteins to conduct structure activity relationship (SAR) studies of the hIL-3 molecule. A further advantage of secretion of (15-125) hIL-3 muteins fused to the lamb signal peptide is that the secreted polypeptide has the correct N-terminal amino acid (Asn) due to the precise nature of the cleavage of the signal peptide by signal peptidase, as part of the secretion process.

The (15-125) hIL-3 muteins of the present invention may include hIL-3 polypeptides having Met-, Ala- or Met-Ala- attached to the N-terminus. When the muteins are expressed in E. coli, polypeptides with and without Met attached to the N-terminus are obtained. The methlonine can in some cases be removed by methionine aminopeptidase.

Amino terminal sequences of some of the hIL-3 muteins made in E. coli were determined using the method described by Hunkapillar et al., (1983). It was found that hIL-3 proteins made in E. coli from genes encoding Met-(15-125) hIL-3 were isolated as Met-(15-125) hIL-3. Proteins produced from genes encoding Met-Ala-(15-125) hIL-3 were produced as Ala-(15-125) hIL-3. The N-termini of proteins made in the cytoplasm of E. coli are affected by posttranslational processing by methionine aminopeptidase (Ben-Bassat et al., 1987) and possibly by other peptidases.

One method of creating the preferred hIL-3 (15-125) mutant genes is cassette mutagenesis [Wells, et al. (1985)] in which a portion of the coding sequence of hIL-3 in a plasmid is replaced with synthetic oligonucleotides that encode the desired amino acid substitutions in a portion of the gene between two restriction sites. In a similar manner amino acid substitutions could be made in the full-length hIL-3 gene, or genes encoding variants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus. When properly assembled these oligonucleotides would encode hIL-3 variants with the desired amino acid substitutions and/or deletions from the N-terminus and/or C-terminus. These and other mutations could be created by those skilled in the art by other mutagenesis methods including; oligonucleotide-directed mutagenesis [Zoller and Smith (1982, 1983, 1984), Smith (1985), Kunkel (1985), Taylor, et al. (1985), Deng and Nickoloff (1992)] or polymerase chain reaction (PCR) techniques [Saiki, (1985)].

Pairs of complementary synthetic oligonucleotides encoding portions of the amino terminus of the hIL-3 gene can be made and annealed to each other. Such pairs would have protruding ends compatible with ligation to NcoI at one end. The NcoI site would include the codon for the initiator methionine. At the other end of oligonucleotide pairs, the protruding (or blunt) ends would be compatible with a restriction site that occurs within the coding sequence of the hIL-3 gene. The DNA sequence of the oligonucleotide would encode sequence for amino acids of hIL-3 with the exception of those substituted and/or deleted from the sequence.

The NcoI enzyme and the other restriction enzymes chosen should have recognition sites that occur only once in the DNA of the plasmid chosen. Plasmid DNA can be treated with the chosen restriction endonucleases then ligated to the annealed oligonucleotides. The ligated mixtures can be used to transform competent JM101 cells to resistance to an appropriate antibiotic. Single colonies can be picked and the plasmid DNA examined by restriction analysis and/or DNA sequencing to identify plasmids with mutant hIL-3 genes.

One example of a restriction enzyme which cleaves within the coding sequence of the hIL-3 gene is ClaI whose recognition site is at codons 20 and 21. The use of ClaI to cleave the sequence of hIL-3 requires that the plasmid DNA be isolated from an E. coli strain that fails to methylate adenines in the DNA at GATC recognition sites. This is because the recognition site for ClaI, ATCGAT, occurs within the sequence GATCGAT which occurs at codons 19, 20 and 21 in the hIL-3 gene. The A in the GATC sequence is methylated in most E. coli host cells. This methylation prevents ClaI from cleaving at that particular sequence. An example of a strain that does not methylate adenines is GM48.

Interpretation of Activity of Single Amino Acid Mutants in IL-3 (15-125)

As illustrated in Tables 6 and 9, there are certain positions in the IL-3 (15-125) molecule which are intolerant of substitutions, in that most or all substitutions at these positions resulted in a considerable decrease in bioactivity. There are two likely classes of such “down-mutations”: mutations that affect overall protein structure, and mutations that interfere directly with the interaction between the IL-3 molecule and its receptor. Mutations affecting the three-dimensional structure of the protein will generally lie in the interior of the protein, while mutations affecting receptor binding will generally lie on the surface of the protein. Although the three-dimensional structure of IL-3 is unknown, there are simple algorithms which can aid in the prediction of the structure. One such algorithm is the use of “helical wheels” (Kaiser, E. T. & Kezdy, F. J., Science, 223:249-255 (1984)). In this method, the presence of alpha helical protein structures can be predicted by virtue of their amphipathic nature. Helices in globular proteins commonly have an exposed hydrophilic side and a buried hydrophobic side. As a broad generalization, in globular proteins, hydrophobic residues are present in the interior of the protein, and hydrophilic residues are present on the surface. By displaying the amino acid sequence of a protein on such a “helical wheel” it is possible to derive a model for which amino acids in alpha helices are exposed and which are buried in the core of the protein. Such an analysis of the IL-3 (15-125) molecule predicts that the following helical residues are buried in the core:

M19, I20, I23, I24, L27, L58, F61, A64, L68, A71, I74, I77, L78, L81, W104, F107, L111, Y114, L115, L118.

In addition, cysteine residues at positions 16 and 84 are linked by a disulfide bond, which is important for the overall structure or “folding” of the protein. Finally, mutations which result in a major disruption of the protein structure may be expressed at low level in the secretion system used in our study, for a variety of reasons: either because the mis-folded protein is poorly recognized by the secretion machinery of the cell; because mis-folding of the protein results in aggregation, and hence the protein cannot be readily extracted from the cells; or because the mis-folded protein is more susceptible to degradation by cellular proteases. Hence, a block in secretion may indicate which positions in the IL-3 molecule which are important for maintenance of correct protein structure.

In order to retain the activity of a variant of IL-3, it is necessary to retain both the structural integrity of the protein, and retain the specific residues important for receptor contact. Hence it is possible to define specific amino acid residues in IL-3 (15-125) which must be retained in order to preserve biological activity.

Residues predicted to be important for interaction with the receptor: D21, E22, E43, D44, L48, R54, R94, D103, K110, F113.

Residues predicted to be structurally important: C16, L58, F61, A64, I74, L78, L81, C84, P86, P92, P96, F107, L111, L115, L118.

The hIL-3 muteins of the present invention may be useful in the treatment of diseases characterized by a decreased levels of either myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells. Among conditions susceptible to treatment with the polypeptides of the present invention is leukopenia, a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, e.g., exposure to chemotherapeutic drugs and of infection or hemorrhage. Therapeutic treatment of leukopenia with these hIL-3 mutant polypeptides of the present invention may avoid undesirable side effects caused by treatment with presently available drugs.

The hIL-3 muteins of the present invention may be useful in the treatment of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia, idiopathic neutropenia, Chdiak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome and myelofibrosis.

Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, and diuretics. The hIL-3 muteins of the present invention may be useful in preventing or treating the bone marrow Suppression or hematopojetic deficiencies which often occur in patients treated with these drugs.

Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis. The hIL-3 muteins of the present invention may be useful in treating such hematopoietic deficiency.

The treatment of hematopoietic deficiency may include administration of the hIL-3 mutein of a pharmaceutical composition containing the hIL-3 mutein to a patient. The hIL-3 muteins of the present invention may also be useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vitro with the muteins of the present invention prior to injecting the cells into a patient.

Various immunodeficiencies e.g., in T and/or B lymphocytes, or immune disorders, e.g., rheumatoid arthritis, may also be beneficially affected by treatment with the hIL-3 mutant polypeptides of the present invention. Immunodeficiencies may be the result of viral infections e.g. HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment. The hIL-3 mutant polypeptides of the present invention may also be employed, alone or in combination with other hematopoietins, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet deficiency), or anemia. Other uses for these novel polypeptides are in the treatment of patients recovering from bone marrow transplants in vivo and ex vivo, and in the development of monoclonal and polyclonal antibodies generated by standard methods for diagnostic or therapeutic use.

Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of one or more of the hIL-3 muteins of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously. When administered, the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.

The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.2-150 μg/kg of non-glycosylated IL-3 protein per kilogram of body weight. This dosage regimen is referenced to a standard level of biological activity which recognizes that native IL-3 generally possesses an EC₅₀ at or about 10 picoMolar to 100 picoMolar in the AML proliferation assay described herein. Therefore, dosages would be adjusted relative to the activity of a given mutein vs. the activity of native (reference) IL-3 and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day. In addition, there may exist specific circumstances where dosages of IL-3 mutein would be adjusted higher or lower than the range of 10-200 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated IL-3 mutein; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, CSF-1, G-CSF, Meg-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, LIF, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, or combinations thereof. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.

Materials and Methods for hTL-1 Mutein Expression in E. coli

Unless noted otherwise, all specialty chemicals were obtained from Sigma Co., (St. Louis, Mo.). Restriction endonucleases, T4 poly-nucleotides kinase, E. coli DNA polymerase I large fragment (Klenow) and T4 DNA ligase were obtained from New England Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.). All chemicals and enzymes were used according to manufacturer's directions.

Escherichia coli Strains

Strain JM101: delta (pro lac), supE, thi, F′ (traD36, proAB, lacI-Q, lacZdeltaM15) (Messing, 1979). This strain can be obtained from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, accession number 33876. MON 105 (W3110 rpoH358) (Obukowicz, et al., 1992) is a derivative of W3110 (Bachmann, 1972) and has been assigned ATCC accession number 55204. Strain GM48: dam-3, dcm-6, gal, ara, lac, thr, leu, tonA, tsx (Marinus, 1973) was used to make plasmid DNA that is not methylated at the sequence GATC.

Genes and Plasmids

The gene used for hIL-3 production in E. coli was obtained from British Biotechnology Incorporated, Cambridge, England, catalogue number BBG14. This gene is carried on a pUC based plasmid designated pP0518. The human IL-3 gene sequence is from Yang, et al. (1986).

The plasmids used for production of hIL-3 in E. coli contain genetic elements whose use has been described (Olins et al., 1988; Olins and Rangwala, 1990). The replicon used is that of pBR327 [(Bolivar et al. (1977); Soberon et al., 1980] which is maintained at a copy number of about 50 in the cell (Covarrubias, et al., (1981)). A gene encoding the beta-lactamase protein is present on the plasmids. This protein confers ampicillin resistance on the cell. This resistance serves as a selectable phenotype for the presence of the plasmid in the cell.

Intracellular Expression Plasmids

For cytoplasmic (intracellular) expression vectors the transcription promoter was derived from the recA gene of E. coli (Sancar et al., 1980). This promoter, designated precA, is contained on 72 base pairs (bp) BglII, BamHI fragment which includes the RNA polymerase binding site and the lexA repressor binding site (the operator). This segment of DNA provides high level transcription that is regulated even when the recA promoter is on a plasmid with the pBR327 origin of replication (Olins et al., 1988) incorporated herein by reference.

Secretion Expression Plasmids

In secretion expression plasmids the transcription promoter was derived from the ara B, A. and D genes of E. coli (Greenfield et al., 1978). This promoter is designated pAraBAD and is contained on a 323 base pair SacrII, BglII restriction fragment. The lamB secretion leader (Wong et al., 1988, Clement et al., 1981) was fused to the N-terminus of the hIL-3 gene at the recognition sequence for the enzyme NcoI (5′CCATGG3′). The hIL-3 genes used were engineered to have a HindIII recognition site (5′AAGCTT3′) following the coding sequence of the gene. Downstream of the gene is a 550 bp fragment containing the origin of replication of the single stranded phage f1 [Olins and Rangwala (1989)].

These hIL-3 variants were expressed as a fusion with the lamB signal peptide operatively joined to the araBAD promoter (Greenfield, 1978) and the g10-L ribosome binding site (Olins et al. 1988). The signal peptide is removed as part of the secretion process. The processed form was selectively released from the periplasm by osmotic shock as a correctly folded and fully active molecule. Secretion of (15-125) hIL-3 was further optimized by using low inducer (arabinose) concentration and by growth at 30° C. These conditions resulted in lower accumulation levels of unprocessed lamB signal peptide (15-125) hIL-3 fusion, maximal accumulation levels of processed (15-125) hIL-3 and selective release of (15-125) hIL-3 by osmotic shock fractionation. The use of a tightly regulated promoter such as araBAD from which the transcription level and hence the expression level can be modulated allowed for the optimization of secretion of (15-125) hIL-3.

The ribosome binding site (RBS) used is that from gene 10 of phage T7 (Olins et al., 1988). This is encoded in a 100 base pair (bp) fragment placed adjacent to precA. In the plasmids used herein, the recognition sequence for the enzyme NcoI (5′CCATGG3′) follows the g10-L RBS. It is at this NcoI site that the hIL-3 genes are joined to the plasmid. It is expected that the nucleotide sequence at this junction will be recognized in mRNA as a functional start site for translation (Olins et al., 1988). The hIL-3 genes used were engineered to have a HindIII recognition site (5′AAGCTT3′) following the coding sequence of the gene. Downstream of the gene is a 550 base pair fragment containing the origin of replication of the single stranded phage f1 (Dente et al., 1983; Olins, et al., 1990) both incorporated herein by reference. A plasmid containing these elements is pMON2341. Another plasmid containing these elements is pMON5847 which has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the accession number ATCC 68912.

Synthesis of Oligonucleotides

oligonucleotides were synthesized by the cyanoethyl method (Addam et al. 1983, McBride, et al. 1983, Sinba et al., 1984) on Nucleotide Synthesizer model 380A or 380B from Applied Biosystems, Inc. (Foster City, Calif.). Some oligonucleotides were purchased from Genosys Biotechnologies Inc. (The Woodlands, Tex.) or Midland Certified Reagent Co. (Midland, Tex.). The degenerate oligonucleotides were synthesized by machine mixing an equal molar ratio of the desired nucleosides in the condensation reaction at degenerate positions. Oligonucleotides were purified by polyacrylamide gel electrophoresis at concentrations from 12-20% (19:1 crosslinked) in 0.5×Tris borate (TBE) buffer (0.045 M Tris, 0.045 M boric acid, 1.25 mM EDTA) as described by Atkinson (1984) . The Oligonucleotides were desalted by passage through a Nensorb 20 column obtained from DuPont/New England Nuclear (Boston, Mass.) using a PREP Automated Sample Processor obtained from DuPont, Co. (Wilmington, Del.).

Quantitation of Synthetic Oligonucleotides

Synthetic oligonucleotides were resuspended in water (100 μl) and quantitated by reading the absorbance at 260 nm on a Beckman DU40 Spectrophotometer (Irvine, Calif.) using a one centimeter by one millimeter quartz cuvette (Maniatis, 1982). The concentration was determined using an extinction coefficient of 1×10⁴ (Voet et al., 1963; Mahler and Cordes, 1966). The oligonucleotide was then diluted to the desired concentration.

Quantitation of synthetic DNA fragments can also be achieved by adding 10 to 100 picomoles of DNA to a solution containing kinase buffer (25 mM Tris pH 8.0, 10 mM MgCl₂, 10 mM DTT and 2 mM spermidine). To the reaction mix is added ATP to 20 micromolar, ATP radiolabeled at the gamma phosphate (5000-10,0000 dpm/pmol) and 5 units of T4 polynucleotide kinase. Radiolabelled material is obtained from New England Nuclear (Boston, Mass.). The 10 microliter mixture is incubated at 37° C. for one hour. A 1 microliter aliquot of the mixture is chromatographed on DEAE paper (DE81 from Whatman) in 0.35 M ammonium bicarbonate. The counts that remain at the origin are used to determine the concentration of the synthetic DNA.

Recombinant DNA Methods

Isolation of plasmid DNA from E. coli cultures was performed as described (Birnboim and Doly, 1979). Some DNAs were purified by Magic™ miniprep columns, available from Promega (Madison, Wis.).

Purified plasmid DNA was treated with restriction endonucleases according to manufacturer's instructions. Analysis of the DNA fragments produced by treatment with restriction enzymes was done by agarose or polyacrylamide gel electrophoresis. Agarose (DNA grade from Fisher, Pittsburgh Pa.) was used at a concentration of 1.0% in a Tris-acetate running buffer (0.04 M Tris-acetate, 0.001M EDTA). Polyacrylamide (BioRad, Richmond Calif.) was used at a concentration of 6% (19:1 crosslinked) in 0.5×Tris-borate buffer (0.045 M Tris, 0.045 M boric acid, 1.25 mM EDTA), hereafter referred to as PAGE.

DNA polymerase I, large fragment, Klenow enzyme was used according to manufacturer's instructions to catalyze the addition of mononucleotides from 5′ to 3′ of DNA fragments which had been treated with restriction enzymes that leave protruding ends. The reactions were incubated at 65° C. for 10 minutes to heat inactivate the Klenow enzyme.

The synthetic oligonucleotides were made without 5′ or 3′ terminal phosphates. In cases where such oligonucleotides were ligated end to end, the oligonucleotides were treated at a concentration of 10 picomoles per microliter with T4 polynucleotide kinase in the following buffer: 25 mM Tris, pH 8.0, 10 mM MgCl₂, 10 mM dithiothreitol, 2 mM spermidine, 1 mM rATP. After incubation for 30 minutes at 37° C., the samples were incubated at 65° C. for five minutes to heat inactivate the kinase.

Synthetic Gene Assembly

The (15-125) hIL-3 gene was divided into four regions separated by five convenient restriction sites. In each of the four regions synthetic oligonucleotides were designed so that they would anneal in complementary pairs, with protruding single stranded ends “or blunt ends” and when the pairs were properly assembled would result in a DNA sequence that encoded a portion of the hIL-3 gene. Amino acid substitutions in the hIL-3 gene were made by designing the oligonucleotides to encode the desired substitutions. The complementary oligonucleotides were annealed at concentration of 1 picomole per microliter in ligation buffer plus 50 mM NaCl. The samples were heated in a 100 ml beaker of boiling water and permitted to cool slowly to room temperature. One picomole of each of the annealed pairs of oligonucleotides were ligated with approximately 0.2 picomoles of plasmid DNA, digested with the appropriate restriction enzymes, in ligation buffer (25 mM Tris pH 8.0, 10 mM MgCl₂, 10 mM dithiothreitol, 1 mM ATP, 2 mM spermidine) with T4 DNA ligase obtained from New England Biolabs (Beverly, Mass.) in a total volume of 20 μl at room temperature overnight.

DNA fragments were isolated from agarose gels by intercepting the restriction fragments on DEAE membranes from Schleicher and Schuell (Keene, N.H.) and eluting the DNA in 10 mM Tris, 1 mM EDTA, 1 M NaCl at 55° C. for 1 hour, according to manufacturer's directions. The solutions containing the DNA fragment were concentrated and desalted by using Centricon 30 concentrators from Amicon (W. R. Grace, Beverly Mass.) according to the manufacturer's directions. Ligations were performed at 15° C. overnight, except as noted, in ligation buffer (66 mM Tris pH 7.5, 6.6 mM MgCl₂, 1 mM dithiothreitol, 0.4 mM ATP) with T₄ ligase obtained from New England Biolabs (Beverly, Mass.).

Polymerase Chain Reaction

Polymerase Chain Reaction (hereafter referred to as PCR) techniques (Saiki, 1985) used the reagent kit and thermal cycler from Perkin-Elmer Cetus (Norwalk, Conn.). PCR is based on a thermostable DNA polymerase from Thermus aquaticus. The PCR technique is a DNA amplification method that mimics the natural DNA replication process in that the number of DNA molecules doubles after each cycle, in a way similar to in vivo replication. The DNA polymerase mediated extension is in a 5′→3′ direction. The term “primer” as used herein refers to an oligonucleotide sequence that provides an end to which the DNA polymerase can add nucleotides that are complementary to a nucleotide sequence. The latter nucleotide sequence is referred to as the “template”, to which the primers are annealed. The amplified PCR product is defined as the region comprised between the 5′ ends of the extension primers. Since the primers have defined sequences, the product will have discrete ends, corresponding to the primer sequenCes. The primer extension reaction was carried out using 20 picomoles (pmoles) of each of the oligonucleotides and 1 picogram of template plasmid DNA for 35 cycles (1 cycle is defined as 94° C. for one minute, 50° C. for two minutes and 72° C. for three minutes). The reaction mixture was extracted with an equal volume of phenol/chloroform (50% phenol and 50% chloroform, volume to volume) to remove proteins. The aqueous phase, containing the amplified DNA, and solvent phase were separated by centrifugation for 5 minutes in a microcentrifuge (Model 5414 Eppendorf Inc, Fremont Calif.). To precipitate the amplified DNA the aqueous phase was removed and transferred to a fresh tube to which was added 1/10 volume of 3M NaOAc (pH 5.2) and 2.5 volumes of ethanol (100% stored at minus 20° C.). The solution was mixed and placed on dry ice for 20 minutes. The DNA was pelleted by centrifugation for 10 minutes in a microcentrifuge and the solution was removed from the pellet. The DNA pellet was washed with 70% ethanol, ethanol removed and dried in a speedvac concentrator (Savant, Farmingdale, N.Y.). The pellet was resuspended in 25 microliters of TE (20 mM Tris-HCl pH 7.9, 1 mM EDTA). Alternatively the DNA was precipitated by adding equal volume of 4M NH₄OAc and one volume of isopropanol [Treco, (1989)]. The solution was mixed and incubated at room temperature for 10 minutes and centrifuged. These conditions selectively precipitate DNA fragments larger than ˜20 bases and were used to remove oligonucleotide primers. One quarter of the reaction was digested with restriction enzymes [Higuchi, (1989)] and on completion heated to 70° C. to inactivate the enzymes.

Two Step Site-directed PCR Mutagenesis

Single amino acid substitution variants were created at positions 17-123 of hIL-3 in two site-directed mutagenesis steps by PCR (Bauer et al. manuscript in preparation).

The single amino acid substitution variants at positions 94-105 of hIL-3 were created as described below. In the first mutagenesis step plasmid DNA, containing the hIL-3 gene (amino acids 15-125), was the template in the PCR reaction. The DNA sequence of one of the oligonucleotide primers was designed to replace 12 base in the hIL-3 gene (15-125) with 12 bases encoding two translation stop codons (5′TAATAA3′), followed. by the recognition sequence (5′GTCGAC3′) restriction enzyme SalI. This 12 base sequence was substituted in the hIL-3 gene following the codon for amino acids 93, 97 and 101. Plasmids containing these mutagenized genes served as the templates for the second mutagenesis step.

In the second mutagenesis step, the 12 base substitution introduced in the first mutagenesis step, was replaced using a 32 fold degenerate oligonucleotide. The degenerate oligonucleotides were synthesized by machine mixing an equal molar ratio of the desired nucleosides in the condensation reaction at degenerate positions. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon. The other bases in the oligonucleotides corresponded to the hIL-3 sequence. The degenerate oligonucleotides theoretically contain 32 different codons, encoding all 20 amino acids and one translation stop codon, at a single position. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. This pool of single amino acid substitutions at a single position is referred to as a “library”. This two step PCR site-directed mutagenesis approach was used to facilitate the identification of single amino acid substitution variants by differential DNA hybridization.

The single amino acid substitution variants at positions 17-93 and 106-123 of hIL-3 (15-125) were created as described below. In the first mutagenesis step plasmid DNA, containing the hIL-3 gene (15-125), was the template in the PCR reaction. The DNA sequence of one of the oligonucleotide primers was designed to delete 18 bases in the hIL-3 gene that encode the following amino acids; 17-22, 23-28, 29-34, 35-40, 41-46, 47-52, 53-58, 59-64, 65-70, 71-76, 77-82, 83-88, 88-93, 106-111, 112-117 and 118-123. Plasmids containing these deletion genes served as the templates for the second mutagenesis step.

In the second mutagenesis step the 18 base deletion, created in the first mutagenesis step, was restored using a 32 fold degenerate oligonucleotide. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon. The other bases in the oligonucleotides corresponded to the hIL-3 sequence. The degenerate oligonucleotides theoretically contain 32 different codons, encoding all 20 amino acids and one translation stop codon, at a single position. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. This pool of single amino acid substitutions at a single position is referred to as a “library”. This two step PCR site-directed mutagenesis approach was used to facilitate the identification of single amino acid substitution variants by differential DNA hybridization.

Recovery of Recombinant Plasmids from Ligation Mixes and Transformation of E. coli Cells with Recombinant Plasmid DNA

E. coli JM101 cells were made competent to take up DNA. Typically, 20 to 100 ml of cells were grown in LB medium to a density of approximately 150 Klett units and then collected by centrifugation. The cells were resuspended in one half culture volume of 50 mM CaCl₂ and held at 4° C. for one hour. The cells were again collected by centrifugation and resuspended in one tenth culture volume of 50 mM CaCl₂. DNA was added to a 150 microliter volume of these cells, and the samples were held at 4° C. for 30 minutes. The samples were shifted to 42° C. for one minute, one milliliter of LB was added, and the samples were shaken at 37° C. for one hour. Cells from these samples were spread on plates containing ampicillin to select for transformants. The plates were incubated overnight at 37° C. Single colonies were picked and grown in LB supplemented with ampicillin overnight at 37° C. with shaking. From these cultures DNA was isolated for restriction analysis.

Typically plasmids were constructed, using methods described herein or by references cited herein, as follows except as noted in examples included herein. DNA fragments were purified from agarose or polyacrylamide gels. Purified DNA fragments were ligated and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin containing plates. Plasmid DNA was isolated from a single colony grown in LB Broth and screened by restriction analysis for the desired construct and sequenced to determine that the DNA sequence was correct.

Culture Media

LB medium (Maniatis et al., 1982) was used for growth of cells for DNA isolation. M9 minimal medium supplemented with 1.0% casamino acids, acid hydrolyzed casein, Difco (Detroit, Mich.) was used for cultures in which recombinant hIL-3 was produced. The ingredients in the M9 medium were as follows: 3g/liter KH₂PO₄, 6 g/l Na₂HPO₄, 0.5 g/l NaCl, 1 g/l NH₄Cl, 1.2 mM MgSO₄, 0.025 mM CaCl₂, 0.2% glucose (0.2% glycerol with the AraBAD promoter), 1% casamino acids, 0.1 ml/l trace minerals (per liter 108 g FeCl₃. 6H₂O, 4.0 g ZnSO₄.7H₂O, 7.0 CoCl₂.2H₂O, 7.0 g Na₂MoO₄.2H₂O, 8.0 g CuSO₄.5H₂O, 2.0 g H₃BO₃, 5.0 g MnSO₄.H₂O, 100 ml concentrated HCl). Bacto agar from Difco was used for solid media and ampicillin (Polycillin-N from Bristol-Meyers, Evansville, Ind.) was added to both liquid and solid LB media at 200 micrograms per milliliter.

DNA Sequence Analysis

The nucleotide sequencing of plasmid DNA was performed using a Genesis 2000 sequencer obtained from DuPont (Wilmington, Del.) according to the methods of Prober et al. (1987) and Sanger et al. (1977). Some DNA sequences were determined using Sequenase™ polymerase according to the protocol of its supplier, U.S. Biochemicals (Cleveland, Ohio).

Production of Recombinant hIL-3 Muteins in E. coli with Vectors Employing the recA Promoter

E. coli strains harboring the plasmids of interest were grown at 37° C. in M9 plus casamino acids medium with shaking in a Gyrotory water bath Model G76 from New Brunswick Scientific (Edison, N.J.). Growth was monitored with a Klett Summerson meter (green 54 filter), Klett Mfg. Co. (New York, N.Y.). At a Klett value of approximately 150, an aliquot of the culture (usually one milliliter) was removed for protein analysis. To the remaining culture, nalidixic acid (10 mg/ml) in 0.1 N NaOH was added to a final concentration of 50 μg/ml. The cultures were shaken at 37° C. for three to four hours after addition of nalidixic acid. A high degree of aeration was maintained throughout the bacterial growth in order to achieve maximal production of the desired gene product. The cells were examined under a light microscope for the presence of retractile bodies (RBs). One milliliter aliquots of the culture were removed for analysis of protein content.

Production of Recombinant hITL-3 Proteins from the AraBAD Promoter in E. coli

E. coli strains harboring the plasmids of interest were grown at 30° C. with shaking in M9 medium plus casamino acids and glycerol. Growth was monitored with a Klett Summerson calorimeter, using a green 54 filter. At a Klett value of about 150, an aliquot of the culture (usually one milliliter) was removed for protein analysis. To the remaining culture, 20% arabinose was added to a final concentration of 0.05%. The cultures were shaken at 30° C. for three to four hours after addition of arabinose. A high degree of aeration was maintained throughout the bacterial growth in order to achieve maximal production of the desired gene product. One milliliter aliquots of the culture were removed for analysis of protein content.

Secretion and Osmotic Shock

Three hour post induction samples were fractionated by osmotic shock [Neu and Heppel (1965)]. The Klett value of the cultures was determined and 1 ml of cells were centrifuged in a Signa mierocentrifuge (West Germany) model 202MK in 1.5 mls snap top microcentrifuge tubes for 5 minutes at 10,000 rpm. The cell pellet was resuspended very gently by pipeting in a room temperature sucrose solution (20% sucrose w/v, 30 mM Tris-Hcl pH7.5, 1 mM EDTA), using 1 μl/1 Klett unit. Following a 10 minute incubation at room temperature, the cells were centrifuged for 5 minutes at 10,000 rpm. The sucrose fraction was carefully removed from the cell pellet. The cell pellet was then resuspended very gently by pipeting in ice cold distilled water, using 1 μl/1 Klett unit. Following a 10 minute incubation on ice, the cells were centrifuged for 5 minutes at 12,000 rpm. The water fraction was carefully removed. Equal volumes of the sucrose and water fractions were pooled and aliquoted to provide samples for ELISA and biological activity screening.

Analysis of Protein Content of E. coli Cultures Producing hTL-3 Mutant Polypeptides

Bacterial cells from cultures treated as described above were collected from the medium by centrifugation. Aliquots of these cells were resuspended in SDS loading. buffer (4×: 6 g SDS, 10 ml beta-mercaptoethanol, 25 ml upper Tris gel stock (0.5 M Tris HCl pH 6.8, 0.4% SDS) brought to 50 ml with glycerol, 0.2% bromophenol blue was added) at a concentration of one microliter per Klett unit. These samples were incubated at 85° C. for five minutes and vortexed. Five or ten microliter aliquots of these samples were loaded on 15% polyacrylamide gels prepared according to the method of Laemmli (1970). Protein bands were visualized by staining the gels with a solution of acetic acid, methanol and water at 5:1:5 (volume to volume) ratio to which Coomassie blue had been added to a final concentration of 1%. After staining, the gels were washed in the same solution without the Coomassie blue and then washed with a solution of 7% acetic acid, 5% methanol. Gels were dried on a gel drier Model SE1160 obtained from Hoeffer (San Francisco, Calif.). The amount of stained protein was measured using a densitometer obtained from Joyce-Loebl (Gateshead, England). The values obtained were a measure of the amount of the stained hIL-3 protein compared to the total of the stained protein of the bacterial cells.

Western Blot Analysis of hTL-3 Muteins Made in E. coli

In some E. coli cultures producing hIL-3, the level of accumulation of the hIL-3 protein is lower than 5% of total bacterial protein. To detect hIL-3 produced at this level, Western blot analysis was used. Proteins from cultures induced with nalidixic acid or arabinose. were run on polyacrylamide gels as described above except that volumes of sample loaded were adjusted to produce appropriate signals. After electrophoresis, the proteins were electroblotted to APT paper, Transa-bind, Schleicher and Schuell (Keene, N.H.) according to the method of Renart et al. (1979). Antisera used to probe these blots had been raised in rabbits, using peptides of the sequence of amino acids 20 to 41 and 94 to 118 of hIL-3 as the immunogens. The presence of bound antibody was detected with Staphylococcal protein A radiolabeled with ¹²⁵I, obtained from New England Nuclear (Boston, Mass.).

Fractionation of E. coli Cells Producing hIL-3 Proteins in the Cytoplasm

Cells from E. coli cultures harboring plasmids that produce hIL-3 muteins were induced with nalidixic acid. After three hours, the hIL-3 muteins accumulated in refractile bodies. The first step in purification of the hIL-3 muteins was to sonivate cells. Aliquots of the culture were resuspended from cell pellets in sonication buffer: 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.1 mM PMSF. These resuspended cells were subjected to several repeated sonication bursts using the microtip from a Sonicator cell disrupter, Model W-375 obtained from Heat Systems-Ultrasonics Inc. (Farmingdale, N.Y.). The extent of sonication was monitored by examining the homogenates under a light microscope. When nearly all of the cells had been broken, the homogenates were fractionated by centrifugation. The pellets, which contain most of the refractile bodies, are highly enriched for hIL-3 muteins.

Methods: Extraction, Refolding and Purification of Interleukin-3 (IL-3) Muteins Expressed as Refractile Bodies in E. coli.

Extraction of refractile bodies (RB's):

For each gram of RB's (and typically one gram is obtained from a 300 ml E. coli culture), 5 ml of a solution containing 6M guanidine hydrochloride (GnHCl), 50 mM 2-N-cyclohexylaminoethanesulfonic acid (CHES) pH 9.5 and 20 mM dithiothreitol (DTT) was added. The RB's were extracted with a Bio-Homogenizer for 15-30 seconds and gently rocked for 2 hours at 5 degrees centigrade (5° C.) to allow the protein to completely reduce and denature.

Refolding of the IL-3 Muteins

The protein solution was transferred to dialysis tubing (1000 molecular weight cut-off) and dialyzed against at least 100 volumes of 4M GnHCl-50 mM CHES pH 8.0. The dialysis was continued overnight at 5° C. while gently stirring. Subsequently dialysis was continued against at least 100 volumes of 2M GnHCl-50 mM CHES pH 8.0 and dialyzed overnight at 5° C. while gently stirring.

Purification of the IL-3 Muteins

The protein solution was removed from the dialysis tubing and acidified by the addition of 40% acetonitrile (CH₃CN)-0.2% trifluoroacetic acid (TFA) to a final concentration of 20% CH₃CN-0.1% TFA. This was centrifuged (16,000×g for 5 minutes) to clarify and the supernatant was loaded onto a Vydac C-18 reversed phase column (10×250 mm) available from Vydac (Hesperia, Calif.) previously equilibrated in 20% CH₃CN-0.1% TFA. The column was eluted with a linear gradient (0.2% CH₃CN/minute) between 40-50% CH₃CN-0.1% TFA at a flow rate of 3 ml/minute while collecting 1.5 ml fractions. The fractions were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and the appropriate fractions pooled. The pooled material was dried by lyophilization or in a Speed Vac concentrator. The dry powder was reconstituted with 10 mM ammonium bicarbonate pH 7.5, centrifuged (16,000×g for 5 minutes) to clarify and assayed for protein concentration by the method of Bradford (1976) with bovine serum albumin as the standard. Such protein can be further analyzed by additional techniques such as, SDS-PAGE, electrospray mass spectrometry, reverse phase HPLC, capillary zone electrophoresis, amino acid composition analysis, and ELISA (enzyme-linked immunosorbent assay).

hIL-3 Sandwich ELISA

IL-3 protein concentrations were determined using a sandwich ELISA based on an affinity purified polyclonal goat anti-rhIL-3. Microtiter plates (Dynatech Immulon II) were coated with 150 μl goat-anti-rhIL-3 at a concentration of approximately 1 μg/ml in 100 mM NaHCO3, pH 8.2. Plates were incubated overnight at room temperature in a chamber maintaining 100% humidity. Wells were emptied and the remaining reactive sites on the plate were blocked with 200 μl of solution containing 10 mM PBS, 3% BSA and 0.05% Tween 20, pH 7.4 for 1 hour at 37° C. and 100% humidity. Wells were emptied and washed 4× with 150 mM NaCl containing 0.05% Tween 20 (wash buffer). Each well then received 150 μl of dilution buffer (10 mM PBS containing 0.1% BSA, 0.01% Tween 20, pH 7.4), containing rhIL-3 standard, control, sample or dilution buffer alone. A standard curve was prepared with concentrations ranging from 0.125 ng/ml to 5 ng/ml using a stock solution of rhIL-3 (concentration determined by amino acid composition analysis). Plates were incubated 2,5 hours at 37° C. and 100% humidity. Wells were emptied and each plate was washed 4× with wash buffer. Each well then received 150 μl of an optimal dilution (as determined in a checkerboard assay format) of goat anti-rhIL-3 conjugated to horseradish peroxidase. Plates were incubated 1.5 hours at 37° C. and 100% humidity. Wells were emptied and each plate was washed 4× with wash buffer. Each well then received 150 ul of ABTS substrate solution (Kirkegaard and Perry). Plates were incubated at room temperature until the color of the standard wells containing 5 ng/ml rhIL-3 had developed enough to yield an absorbance between 0.5-1.0 when read at a test wavelength of 410 nm and a reference wavelength of 570 nm on a Dynatech microtiter plate reader. Concentrations of immunoreactive rhIL-3 in unknown samples were calculated from the standard curve using software supplied with the plate reader.

AML Prolifpraton Assay for Bioactive Human Interleukkin-3

The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, Md.). This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM/CSF supplemented medium (Lange, B., et al., (1987); Valtieri, M., et al., (1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., (1987)). A cell line variant was used, AML 193 1.3, which was adapted for long term growth In IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells were then replated at 1×10⁵ cells/well in a 24 well plate in media containing 100 U/ml IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells were maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.

AML 193 1.3 cells were washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, N.Y.) by centrifuging cell suspensions at 250×g for 10 minutes followed by decantation of supernatant. Pelleted cells were resuspended in HBSS and the procedure was repeated until six wash cycles were completed. Cells washed six times by this procedure were resuspended in tissue culture medium at a density ranging from 2×10⁵ to 5×10⁵ viable cells/ml. This medium was prepared by supplementing Iscove's modified Dulbeccols Medium (IMDM, Hazleton, Lenexa, Kans.) with albumin, transferrin, lipids and 2-mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, Ind.) was added at 500 μg/ml; human transferrin (Boehringer-Mannheim, Indianapolis, Ind.) was added at 100 μg/ml; soybean lipid (Boehringer-Mannheim, Indianapolis, Ind.) was added at 50 μg/ml; and 2-mercaptoethanol (Sigma, St. Louis, Mo.) was added at 5×10⁻⁵M.

Serial dilutions of human interleukin-3 or human interleukin-3 variant protein (hIL-3 mutein) were made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 gl of medium containing interleukin-3 or interleukin-3 variant protein once serial dilutions were completed. Control wells contained tissue culture medium alone (negative control). AMM 193 1.3 cell suspensions prepared as above were added to each well by pipetting 50 μl (2.5×10⁴ cells) into each well. Tissue culture plates were incubated at 37° C. with 5% CO₂ in humidified air for 3 days. On day 3, 0.5 μCi ³H-thymidine (2 Ci/mM, New England Nuclear, Boston, Mass.) was added in 50 μl of tissue culture medium. Cultures were incubated at 37° C. with 5% CO₂ in humidified air for 18-24 hours. Cellular DNA was harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, Md.) using a TOMTEC cell harvester (TOMTEC, Orange, Conn.) which utilized a water wash cycle followed by a 70% ethanol wash cycle. Filter mats were allowed to air dry and then placed into sample bags to which scintillation fluid (Scintiverse II, Fisher Scientific, St. Louis, Mo. or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, Md.) was added. Beta emissions of samples from individual tissue culture wells were counted in a LKB Betaplate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, Md.) and data was expressed as counts per minute of ³H-thymidine incorporated into cells from each tissue culture well. Activity of each human interleukin-3 preparation or human interleukin-3 variant preparation was quantitated by measuring cell proliferation (³H-thymidine incorporation) induced by graded concentrations of interleukin-3 or interleukin-3 variant. Typically, concentration ranges from 0.05 pM-10⁵ pM are quantitated in these assays. Activity is determined by measuring the dose of interleukin-3 or interleukin-3 variant which provides 50% of maximal proliferation [EC₅₀=0.5× (maximum average counts per minute of ³H-thymidine incorporated per well among triplicate cultures of all concentrations of interleukin-3 tested - background proliferation measured by ³H-thymidine incorporation observed in triplicate cultures lacking interleukin-3]. This EC₅₀ value is also equivalent to 1 unit of bioactivity. Every assay was performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.

Relative biological activities of some IL-3 muteins of the present invention are shown in Table 1. The Relative Biological Activity of IL-3 mutants is calculated by dividing the EC₅₀ of (1-133) hIL-3 by the EC₅₀ of the mutant. The Relative Biological Activity may represent the average of replicate assays.

TABLE 1 BIOLOGICAL ACTIVITY OF IL-3 MUTEINS Relative Plasmid Polypeptide Biological Code Structure Activity Reference (1-133)hIL-3 1.0 pMON13286 [SEO ID NO. 69] 8.0 pMON13304 [SEO ID NO. 66] 3.2 * The Relative Biological Activity of IL-3 mutants is calculated by dividing the EC₅₀ of (1-133) hIL-3 by the EC₅₀ of the mutant.

The following assay is used to measure IL-3 mediated sulfidoleukotriene release from human mononuclear cells.

IL-3 Mediated Sulfidoleukotriene Release from Human Mononuclear Cells

Heparin-containing human blood was collected and layered onto an equal volume of Ficoll-Paque (Pharmacia #17-0840-02) ready to use medium (density 1.077 g/ml.). The Ficoll was warmed to room temperature prior to use and clear 50 ml polystyrene tubes were utilized. The Ficoll gradient was spun at 300× g for 30 minutes at room temperature using a H1000B rotor in a Sorvall RT6000B refrigerated centrifuge. The band containing the mononuclear cells was carefully removed, the volume adjusted to 50 mls with Dulbecco's phosphate-buffered saline (Gibco Laboratories cat. #310-4040PK), spun at 400×g for 10 minutes at 40° C. and the supernatant was carefully removed. The cell pellet was washed twice with HA Buffer [20 mM Hepes (Sigma # H-3375), 125 mM NaCl (Fisher # S271-500), 5 mM KCl (sigma # P-9541), 0.5 mM glucose (Sigma # G-5000), 0.025% Human Serum Albumin (Calbiochem #126654) and spun at 300×g, 10 min., 4° C. The cells were resuspended in HACM Buffer (HA buffer supplemented with 1 mM CaC12 (Fisher # C79-500) and 1 mM MgC12 (Fisher # M-33) at a concentration of 1×106 cells/ml and 180 μl were transferred into each well of 96 well tissue culture plates. The cells were allowed to acclimate at 37° C. for 15 minutes. The cells were primed by adding 10 μls of a 20× stock of various concentrations of cytokine to each well (typically 100000, 20000, 4000, 800, 160, 32, 6.4, 1.28, 0 fM IL3). The cells were incubated for 15 minutes at 37° C. Sulfidoleukotriene release was activated by the addition of 10 μl of 20×(1000 nM) fmet-leu-phe (Calbiochem #344252) final concentration 50 nM FMLP and incubated for 10 minutes at 37° C. The plates were spun at 350×g at 4° C. for 20 minutes. The supernatants were removed and assayed for sulfidoleukotrienes using Cayman's Leukotriene C4 EIA kit (Cat. #420211) according to manufacturers' directions. Native (15-125) hIL-3 was run as a standard control in each assay.

Native hIL-3 possesses Considerable inflammatory activity and has been shown to stimulate synthesis of the arachidonic acid metabolites LTC₄, LTD₄, and LTE₄; histamine synthesis and histamine release. Human clinical trials with native hIL-3 have documented inflammatory responses (Biesma, et al., BLOOD, 80:1141-1148 (1992) and Postmus, et al., J. CLIN. ONCOL., 0:1131-1140 (1992)). A recent study indicates that leukotrienes are involved in IL-3 actions in vivo and may contribute significantly to the biological effects of IL-3 treatment (Denzlinger, C., et al., BLOOD, 81:2466-2470 (1993))

Some muteins of the present invention may have an improved therapeutic profile as compared to native hIL-3 or (15-125) hIL-3. For example, some muteins of the present invention may have a similar or more potent growth factor activity relative to native hIL-3 or (15-125) hIL-3 without having a similar or corresponding increase in the stimulation of leukotriene or histamine. These muteins would be expected to have a more favorable therapeutic profile since the amount of polypeptide which needs to be given to achieve the desired growth factor activity (e. g. cell proliferation) would have a lesser leukotriene or histamine stimulating effect. In studies with native hIL-3, the stimulation of inflammatory factors has been an undesirable side effect of the treatment, Reduction or elimination of the stimulation of mediators of inflammation would provide an advantage over the use of native hIL-3.

Some muteins of the present invention may have antigenic profiles which differ from that of native hIL-3. For example, in a competition ELISA with an affinity purified polyclonal goat anti-hIL-3 antibody, native hIL-3 significantly blocked the binding of labeled hIL-3 to polyclonal anti-hIL-3 antibody. Some polypeptides of the present invention, particularly those with several amino acids differing from those of native hIL-3, fail to block the binding of hIL-3 to anti-hIL-3 antibody.

Table 2 lists the sequences of some oligonucleotides used in making the muteins of the present invention.

Table 3 lists the amino acid sequence of native (15-125) hIL-3 (Peptide #1) and the amino acid sequences of some mutant polypeptides of the present invention. The sequences are shown with the amino acid numbering corresponding to that of native hIL-3 [FIG. 1].

TABLE 2 OLIGONUCLEOTIDES Oligo #1 AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGT [SEQ ID NO: 8] AATA Oligo #2 AGCTTATTAC TGTTGAGCCT GCGCGTTCTC CAAGGTTTTC AGATAGAAGG TCAGTTTACG [SEQ ID NO: 9] ACGG Oligo #3 CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG [SEQ ID NO:24] Oligo #4 TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG [SEQ ID NO:25] Oligo #5 CATGGCTAAC TGCTCTAACA TGAT [SEQ ID NO:151] Oligo #6 CGATCAT GTTAGAGCAGTTAGC [SEQ ID NO:152] Oligo #7     IL3MUTNCO TGTCTGCTCA GGCCATGGCT [SEQ ID NO:26] Oligo #8     IL3T93 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGTCGAC TTATTACGTG GGTGCGGCCG [SEQ ID NO:27] TGGCTAG Oligo #9     IL3T97 GCGCGAATTC ATTCCAGTCA CCGTCGACTT ATTAGATTGG ATGTCGCGTG GGTGC [SEQ ID NO:28] Oligo #10    IL3T101 GCGCGAATTC GTCGACTTAT TAGTCCTTGA TATGGATTGG ATG [SEQ ID NO:31] Oligo #11    IL3R94 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG ATGSNNCGTG GGTGCGGCCG [SEQ ID NO:32] TGGCTAG Oligo #12    IL3R95 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG SNNTCGCGTG GGTGCGGCCG [SEQ ID NO:33] TGGC Oligo #13    IL3R96 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATSNNATGTCGCGTG GGTGCGGCCG [SEQ ID NO:34] T Oligo #14    IL3R97 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGSNNTGG ATGTCGCGTG GGTGCGGC [SEQ ID NO:35] Oligo #15    IL3P9497 GATATGGATT GGATGTCGCG TGGG [SEQ ID NO:36] Oligo #16    IL3R98 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TSNNGATTGG ATGTCGCGTG GGTGC [SEQ ID NO:37] Oligo #17    IL3R99 GCGCGAATTC ATTCCAGTCA CCGTCCTTSN NATGGATTGG ATGTCGCGTG GG [SEQ ID NO:38] Oligo #18    IL3R100 GCGCGAATTC ATTCCAGTCA CCGTCSNNGA TATGGATTGG ATGTCGCGT [SEQ ID NO:39] Oligo #19    IL3R101 GCGCGAATTC ATTCCAGTCA CCSNNCTTGA TATGGATTGG ATGTCG [SEQ ID NO:40] Oligo #20    IL3P98100 GTCACCGTCC TTGATATGGA TTGG [SEQ ID NO:41] Oligo #21    IL3R102 GCGCGAATTC ATTCCAGTCS NNGTCCTTGA TATGGATTGG ATG [SEQ ID NO:42] Oligo #22    IL3R103 GCGCGAATTC ATTCCASNNA CCGTCCTTGA TATGGATTGG [SEQ ID NO:43] Oligo #23    IL3R104 GCGCGAATTC ATTSNNGTCA CCGTCCTTGA TATGGAT [SEQ ID NO:44] Oligo #24    IL3R105 GCGCGAATTC SNNCCAGTCA CCGTCCTTGA TATG [SEQ ID NO:45] Oligo #25    IL3P102105 GAATTCATTC CAGTCACCGT TCCTT [SEQ ID NO:46] Oligo #26    IL3MUTR1 CGCGCGGAAT TCATTCCAGT CACCGT [SEQ ID NO:47] Oligo #27    DEL1722 CGCGCGCCAT GGCTAACTGC ATTATAACAC ACACTTAAAG CA [SEQ ID NO:48] Oligo #28    DEL2328 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAACA GCCACCTTTG CCTTTGCT [SEQ ID NO:49] Oligo #29    DEL2934 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCTGG [SEQ ID NO:50] ACTTCAACAA CCTCAA Oligo #30    DEL3540 GCGCGCGATA TCTTGGTCTT CTTCACCATT CAGCGGCAGC GGTGGCTGCT [SEQ ID NO:51] Oligo #31    DEL4146 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATGAGGT TGTTGAAGTC [SEQ ID NO:52] CAGCA Oligo #32    DEL4752 GCGCGCCTCG AGGTTTGGAC GACGAAGATC TTGGTCTTCA CCATTGA [SEQ ID NO:53] Oligo #33    DEL5358 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGT TATTTTCCAT [SEQ ID NO:54] CAGGATAT Oligo #34    DEL5964 GCGCGCTGAT GCATTCTGCA GAGACTTGAC GAGGTTTGGA CGACGAAGGT [SEQ ID NO:55] Oligo #35    DEL6570 GCGCGCCTCG AGGCATTCAA CCGTGCTGCA TCAGCAATTG AGAGCAT [SEQ ID NO:56] Oligo #36    DEL7176 GCGCGCCTGC AGAATATTCT TAAAAATCTC CTGCC [SEQ ID NO:57] Oligo #37    DEL7782 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCCCATGTC TGCCGCTAGC CAC [SEQ ID NO:58] Oligo #38    DEL8388 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GACGGCCGCA [SEQ ID NO:59] CCCACGCGAC A Oligo #39    DEL8893 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCA GGGCAGACAT [SEQ ID NO:60] GGCAGGA Oligo #40    DEL106111 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTAT [SEQ ID NO:61] TCCAGTCACC GTCCTTGA Oligo #41    DEL112117 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA CAGTTTACGA CGGAATTCAT [SEQ ID NO:62] Oligo #42    DEL118123 CGCGCGAAGC TTATTACTGT TGGGTTTTCA GATAGAAGGT CA [SEQ ID NO:63] Oligo #43    R17IL3    Length: 000058 CGCGCGCCAT GGCTAACTGC NNSAACATGA TCGATGAAAT TATAACACAC TTAAAGCA [SEQ ID NO:64] Oligo #44    R18IL3    Length: 000058 CGCGCGCCAT GGCTAACTGC TCTNNSATGA TCGATGAAAT TATAACACAC TTAAAGCA [SEQ ID NO:222] Oligo #45    R19IL3    Length: 000058 CGCGCGCCAT GGCTAACTGC TCTAACNNSA TCGATGAAAT TATAACACAC TTAAAGCA [SEQ ID NO:223] Oligo #46    R20IL3    Length: 000058 CGCGCGCCAT GGCTAACTGC TCTAACATGN NSGATGAAAT TATAACACAC TTAAAGCA [SEQ ID NO:224] Oligo #47    R21IL3    Length: 000058 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCNNSGAAAT TATAACACAC TTAAAGCA [SEQ ID NO:225] Oligo #48    R22IL3    Length: 000058 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATNNSAT TATAACACAC TTAAAGCA [SEQ ID NO:226] Oligo #49    R23IL3    Length: 000076 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAANN SATAACACAC TTAAAGCAGC [SEQ ID NO:227] CACCTTTGCC TTTGCT Oligo #50    R24IL3    Length: 000076 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TNNSACACAC TTAAAGCAGC [SEQ ID NO:228] CACCTTTGCC TTTGCT Oligo #51    R25IL3    Length: 000076 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATANNSCAC TTAAAGCAGC [SEQ ID NO:229] CACCTTTGCC TTTGCT Oligo #52    R26IL3    Length: 000076 CGCGCGCCAT GGCTAACTCC TCTAACATGA TCGATGAAAT TATAACANNS TTAAAGCAGC [SEQ ID NO:74] CACCTTTGCC TTTGCT Oligo #53    R27IL3    Length: 000076 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC NNSAAGCAGC [SEQ ID NO:75] CACCTTTGCC TTTGCT Oligo #54    R28IL3    Length: 000076 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTANNSCAGC [SEQ ID NO:76] CACCTTTGCC TTTGCT Oligo #55    R29IL3    Length: 000094 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGNNSC [SEQ ID NO:77] CACCTTTGCC TTTGCTGGAC TTCAACAACC TCAA Oligo #56    R30IL3    Length: 000094 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGN [SEQ ID NO:78] NSCCTTTGCC TTTGCTGGAC TTCAACAACC TCAA Oligo #57    R31IL3    Length: 000094 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC [SEQ ID NO:79] CANNSTTGCC TTTGCTGGAC TTCAACAACC TCAA Oligo #58    R32IL3    Length: 000094 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC [SEQ ID NO:80] CACCTNNSCC TTTGCTGGAC TTCAACAACC TCAA Oligo #59    R33IL3    Length: 000094 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC [SEQ ID NO:81] CACCTTTGNN STTGCTGGAC TTCAACAACC TCAA Oligo #60    R34IL3    Length: 000094 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC [SEQ ID NO:82] CACCTTTGCC TNNSCTGGAC TTCAACAACC TCAA Oligo #61    R35IL3    Length: 000065 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAG TCSNNCAGCG GCAGCGGTGG [SEQ ID NO:83] CTGCT Oligo #62    R36IL3    Length: 000065 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAS NNCAGCAGCG GCAGCG [SEQ ID NO:84] GTGGCTGCT Oligo #63    R37IL3    Length: 000065 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTSNNG TCCAGCAGCG GCAGCGGTGG [SEQ ID NO:85] CTGCT Oligo #64    R38IL3    Length: 000065 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTSNNGAAG TCCAGCAGCG GCAGCGGTGG [SEQ ID NO:86] CTGCT Oligo #65    R39IL3    Length: 000065 GCGCGCGATA TCTTGGTCTT CACCATTGAG SNNGTTGAAG TCCAGCAGCG GCAGCGGTGG [SEQ ID NO:87] CTGCT Oligo #66    R40IL3    Length: 000065 GCGCGCGATA TCTTGGTCTT CACCATTSNN GTTGTTGAAG TCCAGCAGCG GCAGCGGTGG [SEQ ID NO:88] CTGCT Oligo #67    R41IL3    Length: 000083 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCACC [SEQ ID NO:89] SNNGAGGTTG TTGAAGTCCA GCA Oligo #68    R42IL3    Length: 000083 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCSNN [SEQ ID NO:90] ATTGAGGTTG TTGAAGTCCA GCA Oligo #69    R43IL3    Length: 000083 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCSNNACC [SEQ ID NO:91] ATTGAGGTTG TTGAAGTCCA GCA Oligo #70    R44IL3    Length: 000083 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GSNNTTCACC [SEQ ID NO:92] ATTGAGGTTG TTGAAGTCCA GCA Oligo #71    R45IL3    Length: 000083 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCSN NGTCTTCACC [SEQ ID NO:93] ATTGAGGTTG TTGAAGTCCA GCA Oligo #72    R46IL3    Length: 000083 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATSNNTT GGTCTTCACC [SEQ ID NO:94] ATTGAGGTTG TTGAAGTCCA GCA Oligo #73    R47IL3    Length: 000065 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGSNNATCTT GGTCTTCACC [SEQ ID NO:95] ATTGA Oligo #74    R48IL3    Length: 000065 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATS NNGATATCTT GGTCTTCACC [SEQ ID NO:96] ATTGA Oligo #75    R49IL3    Length: 000065 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCSNNC AGGATATCTT GGTCTTCACC [SEQ ID NO:97] ATTGA Oligo #76    R50IL3    Length: 000065 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTSNNCATC AGGATATCTT GGTCTTCACC [SEQ ID NO:98] ATTGA Oligo #77    R51IL3    Length: 000065 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT SNNTTCCATC AGGATATCTT GGTCTTCACC [SEQ ID NO:99] ATTGA Oligo #78    R52IL3    Length: 000065 GCGCGCCTCG AGGTTTGGAC GACGAAGSNN ATTTTCCATC AGGATATCTT GGTCTTCACC [SEQ ID NO:100] ATTGA Oligo #79    R53IL3    Length: 000086 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG [SEQ ID NO:101] ACGSNNGTTA TTTTCCATCA GGATAT Oligo #80    R54IL3    Length: 000086 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG [SEQ ID NO:102] SNNAAGGTTA TTTTCCATCA GGATAT Oligo #81    R55IL3    Length: 000086 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGSNN [SEQ ID NO:103] ACGAAGGTTA TTTTCCATCA GGATAT Oligo #82    R56IL3    Length: 000086 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTSNNACG [SEQ ID NO:104] ACGAAGGTTA TTTTCCATCA GGATAT Oligo #83    R57IL3    Length: 000086 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GSNNTGGACG [SEQ ID NO:105] ACGAAGGTTA TTTTCCATCA GGATAT Oligo #84    R58IL3    Length: 000086 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCSN NGTTTGGACG [SEQ ID NO:106] ACGAAGGTTA TTTTCCATCA GGATAT Oligo #85    R59IL3    Length: 000068 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCSNNGA GGTTTGGACG [SEQ ID NO:107] ACGAAGGT Oligo #86    R60IL3    Length: 000068 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AASNNCTCGA GGTTTGGACG [SEQ ID NO:108] ACGAAGGT Oligo #87    R61IL3    Length: 000068 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTS NNTGCCTCGA GGTTTGGACG [SEQ ID NO:109] ACGAAGGT Oligo #88    R62IL3    Length: 000068 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGSNNG AATGCCTCGA GGTTTGGACG [SEQ ID NO:110] ACGAAGGT Oligo #89    R63IL3    Length: 000068 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCSNNGTTG AATGCCTCGA GGTTTGGACG [SEQ ID NO:111] ACGAAGGT Oligo #90    R64IL3    Length: 000068 GCGCGCTGAT GCATTCTGCA GAGACTTGAC SNNACGGTTG AATGCCTCGA GGTTTGGACG [SEQ ID NO:112] ACGAAGGT Oligo #91    R65IL3    Length: 000065 GCGCGCCTCG AGGCATTCAA CCGTGCTNNS AAGTCTCTGC AGAATGCATC AGCAATTGAG [SEQ ID NO:113] AGCAT Oligo #92    R66IL3    Length: 000065 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC NNSTCTCTGC AGAATGCATC AGCAATTGAG [SEQ ID NO:114] AGCAT Oligo #93    R67IL3    Length: 000065 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGNNSCTGC AGAATGCATC AGCAATTGAG [SEQ ID NO:115] AGCAT Oligo #94    R68IL3    Length: 000065 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTNNSC AGAATGCATC AGCAATTGAG [SEQ ID NO:116] AGCAT Oligo #95    R69IL3    Length: 000065 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGN NSAATGCATC AGCAATTGAG [SEQ ID NO:117] AGCAT Oligo #96    R70IL3    Length: 000065 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGC AGNNSGCATC AGCAATTGAG [SEQ ID NO:118] AGCAT Oligo #97    R71IL3    Length: 000053 GCGCGCCTGC AGAATNNSTC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCC [SEQ ID NO:119] Oligo #98    R72IL3    Length: 000053 GCGCGCCTGC AGAATGCANN SGCAATTGAG AGCATTCTTA AAAATCTCCT GCC [SEQ ID NO:120] Oligo #99    R73IL3    Length: 000053 GCGCGCCTGC AGAATGCATC ANNSATTGAG AGCATTCTTA AAAATCTCCT GCC [SEQ ID NO:121] Oligo #100   R74IL3    Length: 000053 GCGCGCCTGC AGAATGCATC AGCANNSGAG AGCATTCTTA AAAATCTCCT GCC [SEQ ID NO:122] Oligo #101   R75IL3    Length: 000053 GCGCGCCTGC AGAATGCATC AGCAATTNNS AGCATTCTTA AAAATCTCCT GCC [SEQ ID NO:123] Oligo #102   R76IL3    Length: 000053 GCGCGCCTGC AGAATGCATC AGCAATTGAG NNSATTCTTA AAAATCTCCT GCC [SEQ ID NO:124] Oligo #103   R77IL3    Length: 000071 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCNNSCTTA AAAATCTCCT GCCATGTCTG [SEQ ID NO:125] CCGCTAGCCA C Oligo #104   R78IL3    Length: 000071 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTNNSA AAAATCTCCT GCCATGTCTG [SEQ ID NO:126] CCGCTAGCCA C Oligo #105   R79IL3    Length: 000071 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTN NSAATCTCCT GCCATGTCTG [SEQ ID NO:127] CCGCTAGCCA C Oligo #106   R80IL3    Length: 000071 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AANNSCTCCT GCCATGTCTG [SEQ ID NO:138] CCGCTAGCCA C Oligo #107   R81IL3    Length: 000071 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATNNSCT GCCATGTCTG [SEQ ID NO:139] CCGCTAGCCA C Oligo #108   R82IL3    Length: 000071 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCNN SCCATGTCTG [SEQ ID NO:140] CCGCTAGCCA C Oligo #109   R83IL3    Length: 000089 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GNNSTGTCTG [SEQ ID NO:141] CCGCTAGCCA CGGCCGCACC CACGCGACA Oligo #110   R84IL3    Length: 000089 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCANNSCTG [SEQ ID NO:142] CCGCTAGCCA CGGCCGCACC CACGCGACA Oligo #111   R85IL3    Length: 000089 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTNNS [SEQ ID NO:143] CCGCTAGCCA CGGCCGCACC CACGCGACA Oligo #112   R86IL3    Length: 000089 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG [SEQ ID NO:157] NNSCTAGCCA CGGCCGCACC CACGCGACA Oligo #113   R87IL3    Length: 000089 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG [SEQ ID NO:158] CCGNNSGCCA CGGCCGCACC CACGCGACA Oligo #114   R88IL3    Length: 000089 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG [SEQ ID NO:159] CCGCTANNSA CGGCCGCACC CACGCGACA Oligo #115   R89IL3    Length: 000086 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCGGC [SEQ ID NO:160] SNNGGCCAGG GCCAGACATG GCAGGA Oligo #116   R90IL3    Length: 000086 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCSNN [SEQ ID NO:161] CGTGGCCAGG GGCAGACATG GCAGGA Oligo #117   R91IL3    Length: 000086 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGSNNGGC [SEQ ID NO:162] CGTGGCCAGG GGCAGACATG GCAGGA Oligo #118   R92IL3    Length: 000086 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TSNNTGCGGC [SEQ ID NO:163] CGTGGCCAGG GGCAGACATG GCAGGA Oligo #119   R93IL3    Length: 000086 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGSN NGGGTGCGGC [SEQ ID NO:164] CGTGGCCAGG GGCAGACATG GCAGGA Oligo #120   3PR106    Length: 000048 TTTCAGATAG AAGGTCAGTT TACGACGGAA SNNATTCCAG TCACCGTC [SEQ ID NO:165] Oligo #121   3PR107    Length: 000048 TTTCAGATAG AAGGTCAGTT TACGACGSNN TTCATTCCAG TCACCGTC [SEQ ID NO:166] Oligo #122   3PR108    Length: 000048 TTTCAGATAG AAGGTCAGTT TACGSNNGAA TTCATTCCAG TCACCGTC [SEQ ID NO:167] Oligo #123   3PR109    Length: 000048 TTTCAGATAG AAGGTCAGTT TSNNACGGAA TTCATTCCAG TCACCGTC [SEQ ID NO:168] Oligo #124   3PR110    Length: 000048 TTTCAGATAG AAGGTCAGSN NACGACGGAA TTCATTCCAG TCACCGTC [SEQ ID NO:169] Oligo #125   3PR111    Length: 000048 TTTCAGATAG AAGGTSNNTT TACGACGGAA TTCATTCCAG TCACCGTC [SEQ ID NO:170] Oligo #126   IL3MUTD3  Length: 000023 CGCGCGAAGC TTATTACTGT TGA [SEQ ID NO:171] Oligo #127   R112IL3   Length: 000078 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAASNNCA [SEQ ID NO:172] GTTTACGACG GAATTCAT Oligo #128   R113IL3   Length: 000078 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TASNNGGTCA [SEQ ID NO:173] GTTTACGACG GAATTCAT Oligo #129   R114IL3   Length: 000078 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGS NNGAAGGTCA [SEQ ID NO:174] GTTTACGACG GAATTCAT Oligo #130   R115IL3   Length: 000078 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTSNNA TAGAAGGTCA [SEQ ID NO:175] GTTTACGACG GAATTCAT Oligo #131   R116IL3   Length: 000078 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTSNNCAGA TAGAAGGTCA [SEQ ID NO:176] GTTTACGACG GAATTCAT Oligo #132   R117IL3   Length: 000078 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA SNNTTTCAGA TAGAAGGTCA [SEQ ID NO:177] GTTTACGACG GAATTCAT Oligo #133   R118IL3   Length: 000060 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCSNN GGTTTTCAGA TAGAAGGTCA [SEQ ID NO:178] Oligo #134   R119IL3   Length: 000060 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTSNNCAA GGTTTTCAGA TAGAAGGTCA [SEQ ID NO:179] Oligo #135   R120IL3   Length: 000060 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CSNNCTCCAA GGTTTTCAGA TAGAAGGTCA [SEQ ID NO:180] Oligo #136   R121IL3   Length: 000060 CGCGCGAAGC TTATTACTGT TGAGCCTGSN NGTTCTCCAA GGTTTTCAGA TAGAAGGTCA [SEQ ID NO:181] Oligo #137   R122IL3   Length: 000060 CGCGCGAAGC TTATTACTGT TGAGCSNNCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA [SEQ ID NO:182] Oligo #138   R123IL3   Length: 000060 CGCGCGAAGC TTATTACTGT TGSNNCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA [SEQ ID NO:183] Oligo #139   P1722IL3  Length: 000024 TGCTCTAACA TGATCGATGA AATT [SEQ ID NO:184] Oligo #140   P2328IL3  Length: 000024 GAAATTATAA CACACTTAAA GCAG [SEQ ID NO:185] Oligo #141   P2934IL3  Length: 000024 AAGCAGCCAC CTTTGCCTTT GCTG [SEQ ID NO:186] Oligo #142   P3540IL3  Length: 000024 AAGCAGCCAC CGCTGCCGCT GCTG [SEQ ID NO:187] Oligo #143   PRB41-46  Length: 000024 CTCAATGGTG AAGACCAAGA TATC [SEQ ID NO:188] Oligo #144   PRB47-52  Length: 000024 GATATCCTGA TGGAAAATAA CCTT [SEQ ID NO:189] Oligo #145   PRB53-58  Length: 000024 AACCTTCGTC GTCCAAACCT CGAG [SEQ ID NO:190] Oligo #146   PRB59-64  Length: 000024 CTCGAGGCAT TCAACCGTGC TGTC [SEQ ID NO:191] Oligo #147   PRB65-70  Length: 000024 GCTGTCAAGT CTCTGCAGAA TGCA [SEQ ID NO:192] Oligo #148   P7176IL3  Length: 000024 AATGCATCAG CAATTGAGAG CATT [SEQ ID NO:193] Oligo #149   P7782IL3  Length: 000024 AGCATTCTTA AAAATCTCCT GCCA [SEQ ID NO:194] Oligo #150   P8388IL3  Length: 000024 CTGCCATGTC TGCCCCTGGC CACG [SEQ ID NO:195] Oligo #151   P8893IL3  Length: 000024 CTGGCCACGG CCGCACCCAC GCGA [SEQ ID NO:196] Oligo #152   P106111   Length: 000024 AATGAATTCC GTCGTAAACT GACC [SEQ ID NO:197] Oligo #153   P112117   Length: 000024 CTGACCTTCT ATCTGAAAAC CTTG [SEQ ID NO:198] Oligo #154   P118123   Length: 000024 ACCTTGGAGA ACGCGCAGGC TCAA [SEQ ID NO:199] Oligo #155   PSTECRI1.REQ  Length: 000022 GAATGCATCA GCAATTGAGA GC [SEQ ID NO:200] Oligo #156   PSTECRI5.REQ  Length: 000020 AATTGCTGAT GCATTCTGCA [SEQ ID NO:201] Oligo #157   PSTECRI2.REQ  Length: 000024 ATTCTTAAAA ATCTCCTGCC ATGT [SEQ ID NO:202] Oligo #158   PSTECRI6.REQ  Length: 000024 CAGGAGATTT TTAAGAATGC TCTC [SEQ ID NO:203] Oligo #159   PSTECRI3.REQ  Length: 000030 CTGCCCCTGG CCACGGCCGC ACCCACGCGA [SEQ ID NO:204] Oligo #160   PSTECRI7.REQ  Length: 000030 GGGTGCGGCC GTGGCCAGGG GCAGACATGG [SEQ ID NO:205] Oligo #161   98I100R4.REQ  Length: 000034 CATCCAATCA TCATCCGTGA CGGTGACTGG AATG [SEQ ID No:206] Oligo #162   98I100R8.REQ  Length: 000044 AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGATGTC GCGT [SEQ ID NO:207] Oligo 163   95R8I0R4.REQ  Length: 000034 CGCCCAATCA TCATCCGTGA CGGTGACTGG AATG [SEQ ID NO:208] Oligo #164   95R8I0R8.REQ  Length: 000044 AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGGCGTC GCGT [SEQ ID NO:209] Oligo #165   NCOECRV1.REQ  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA [SEQ ID NO:210] Oligo #166   NCOECRV4.REQ  Length: 000045 CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC [SEQ ID NO:211] Oligo #167   NCOECRV2.REQ  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC [SEQ ID NO:212] Oligo #168   NCOECRV5.REQ  Length: 000036 GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG [SEQ ID NO:213] Oligo #169   2D5M6SUP.REQ  Length: 000027 AACAACCTCA ATGACGAAGA CATGTCT [SEQ ID NO:214] Oligo #170   2D5M6SLO.REQ  Length: 000018 AGACATGTCT TCGTCATT [SEQ ID NO:215] Oligo #15(A)   Length: 000016           TGAACCATAT GTCAGG [SEQ ID NO:29] Oligo #16(A)   Length: 000024           AATTCCTGAC ATATGGTTCA TGCA [SEQ ID NO:30] Oligo #51(A)   Length: 000034 GCCGATACCGCGGCATACTCCCACCATTCAGAGA [SEQ ID NO:155] Oligo #52(A)   Length: 000033 GCCGATAAGATCTAAAACGGGTATGGAGAAACA [SEQ ID NO:156] Oligo #171   Length: 000040 CATGGCTAAC TGCTCTAACA TGATCAACGA AATTATAACA [SEQ. ID NO:69] Oligo #172  Length: 000045 CTTTAAGTGT GTTATAATTT CGTTGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:70] Oligo #173  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCCAAGA AATTATAACA [SEQ. ID NO:71] Oligo #174  Length: 000045 CTTTAAGTGT GTTATAATTT CTTGGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:72] Oligo #175  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGAAGA AATTATAACA [SEQ. ID NO:73] Oligo #176  Length: 000045 CTTTAAGTGT GTTATAATTT CTTCGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:219] Oligo #177  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCAGCGA AATTATAACA [SEQ. ID NO:230] Oligo #178  Length: 000045 CTTTAAGTGT GTTATAATTT CGCTGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:231] Oligo #179  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCACCGA AATTATAACA [SEQ. ID NO:232] Oligo #180  Length: 000045 CTTTAAGTGT GTTATAATTT CCGTGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:233] Oligo #181  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGATAA CATTATAACA [SEQ. ID NO:234] Oligo #182  Length: 000045 CTTTAAGTGT GTTATAATGT TATCGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:235] Oligo #183  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGATGA CATTATAACA [SEQ. ID NO:236] Oligo #184  Length: 000045 CTTTAAGTGT GTTATAATGT CATCGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:237] Oligo #185  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGATCA GATTATAACA [SEQ. ID NO:238] Oligo #186  Length: 000045 CTTTAAGTGT GTTATAATCT GATCGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:239] Oligo #187  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGATCT GATTATAACA [SEQ. ID NO:240] Oligo #188  Length: 000045 CTTTAAGTGT GTTATAATCA GATCGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:241] Oligo #189  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGATGT TATTATAACA [SEQ. ID NO:242] Oligo #190  Length: 000045 CTTTAAGTGT GTTATAATAA CATCGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:243] Oligo #191  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTGCTCTG GACTTC [SEQ. ID NO:244] Oligo #192  Length: 000036 GAGGTTGTTG AAGTCCAGAG CAGGCAAAGG TGGCTG [SEQ. ID NO:245] Oligo #193  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTCGTCTG GACTTC [SEQ. ID NO:246] Oligo #194  Length: 000036 GAGGTTGTTG AAGTCCAGAC GAGGCAAAGG TGGCTG [SEQ. ID NO:247] Oligo #195  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTCAGCTG GACTTC [SEQ. ID NO:248] Oligo #196  Length: 000036 GAGGTTGTTG AAGTCCAGCT GAGGCAAAGG TGGCTG [SEQ. ID NO:249] Oligo #197  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTGAACTG GACTTC [SEQ. ID NO:250] Oligo #198  Length: 000036 GAGGTTGTTG AAGTCCAGCT CAGGCAAAGG TGGCTG [SEQ. ID NO:251] Oligo #199  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTATCCTG GACTTC [SEQ. ID NO:252] Oligo #200  Length: 000036 GAGGTTGTTG AAGTCCAGGA TAGGCAAAGG TGGCTG [SEQ. ID NO:253] Oligo #201  Length: 000036 CACTTAAAGC AGCCACCTTT CCCTTTCCTG GACTTC [SEQ. ID NO:254] Oligo #202  Length: 000036 GAGGTTGTTG AAGTCCAGGA AAGGCAAAGG TGGCTG [SEQ. ID NO:255] Oligo #203  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTACCCTG GACTTC [SEQ. ID NO:256] Oligo #204  Length: 000036 GAGGTTGTTG AAGTCCAGGG TAGGCAAAGG TGGCTG [SEQ. ID NO:257] Oligo #205  Length: 000027 AACAACCTCA ATCGTGAAGA CCAAGAT [SEQ. ID NO:258] Oligo #206  Length: 000018 ATCTTGGTCT TCACGATT [SEQ. ID NO:259] Oligo #207  Length: 000027 AACAACCTCA ATAACGAAGA CCAAGAT [SEQ. ID NO:260] Oligo #208  Length: 000018 ATCTTGGTCT TCGTTATT [SEQ. ID NO:261] Oligo #209  Length: 000027 AACAACCTCA ATGAAGAAGA CCAAGAT [SEQ. ID NO:262] Oligo #210  Length: 000018 ATCTTGGTCT TCTTCATT [SEQ. ID NO:263] Oligo #211  Length: 000027 AACAACCTCA ATATCGAAGA CCAAGAT [SEQ. ID NO:264] Oligo #212  Length: 000018 ATCTTGGTCT TCGATATT [SEQ. ID NO:265] Oligo #213  Length: 000027 AACAACCTCA ATCTGGAAGA CCAAGAT [SEQ. ID NO:266] Oligo #214  Length: 000018 ATCTTGGTCT TCCAGATT [SEQ. ID NO:267] Oligo #215  Length: 000027 AACAACCTCA ATAAAGAAGA CCAAGAT [SEQ. ID NO:268] Oligo #216  Length: 000018 ATCTTGGTCT TCTTTATT [SEQ. ID NO:269] Oligo #217  Length: 000027 AACAACCTCA ATATGGAAGA CCAAGAT [SEQ. ID NO:270] Oligo #218  Length: 000018 ATCTTGGTCT TCCATATT [SEQ. ID NO:271] Oligo #219  Length: 000027 AACAACCTCA ATTTCGAAGA CCAAGAT [SEQ. ID NO:272] Oligo #220  Length: 000018 ATCTTGGTCT TCGAAATT [SEQ. ID NO:273] Oligo #221  Length: 000027 AACAACCTCA ATACCGAAGA CCAAGAT [SEQ. ID NO:274] Oligo #222  Length: 000018 ATCTTGGTCT TCGGTATT [SEQ. ID NO:275] Oligo #223  Length: 000027 AACAACCTCA ATTACGAAGA CCAAGAT [SEQ. ID. NO:276] Oligo #224  Length: 000018 ATCTTGGTCT TCGTAATT [SEQ. ID NO:277] Oligo #225  Length: 000027 AACAACCTCA ATGTTGAAGA CCAAGAT [SEQ. ID NO:278] Oligo #226  Length: 000018 ATCTTGGTCT TCAACATT [SEQ. ID NO:279] Oligo #227  Length: 000027 AACAACCTCA ATGGGCGTGA CCAAGAT [SEQ. ID NO:280] Oligo #228  Length: 000018 ATCTTGGTCT CGCCCATT [SEQ. ID NO:281] Oligo #229  Length: 000027 AACAACCTCA ATGGGCAGGA CCAAGAT [SEQ. ID NO:282] Oligo #230  Length: 000018 ATCTTGGTCC TGCCCATT [SEQ. ID NO:283] Oligo #231  Length: 000027 AACAACCTCA ATGGGGGTGA CCAAGAT [SEQ. ID NO:284] Oligo #232  Length: 000018 ATCTTGGTCA CCCCCATT [SEQ. ID NO:285] Oligo #233  Length: 000027 AACAACCTCA ATGGGACCGA CCAAGAT [SEQ. ID NO:286] Oligo #234  Length: 000018 ATCTTGGTCG GTCCCATT [SEQ. ID NO:287] Oligo #235  Length: 000027 AACAACCTCA ATGGGGAAGC TCAAGAT [SEQ. ID NO:288] Oligo #236  Length: 000018 ATCTTGAGCT TCCCCATT [SEQ. ID NO:289] Oligo #237  Length: 000027 AACAACCTCA ATGGGGAAAA CCAAGAT [SEQ. ID NO:290] Oligo #238  Length: 000018 ATCTTGGTTT TCCCCATT [SEQ. ID NO:291] Oligo #239  Length: 000027 AACAACCTCA ATGGGGAACA GCAAGAT [SEQ. ID NO:292] Oligo #240  Length: 000018 ATCTTGCTGT TCCCCATT [SEQ. ID NO:293] Oligo #241  Length: 000027 AACAACCTCA ATGGGGAAGA ACAAGAT [SEQ. ID NO:294] Oligo #242  Length: 000018 ATCTTGTTCT TCCCCATT [SEQ. ID NO:295] Oligo #243  Length: 000027 AACAACCTCA ATGGGGAAGA CGCTGAT [SEQ. ID NO:296] Oligo #244  Length: 000018 ATCAGCGTCT TCCCCATT [SEQ. ID NO:297] Oligo #245  Length: 000027 AACAACCTCA ATGGGGAAGA CCGTGAT [SEQ. ID NO:298] Oligo #246  Length: 000018 ATCACGGTCT TCCCCATT [SEQ. ID NO:299] Oligo #247  Length: 000027 AACAACCTCA ATGGGGAAGA CAACGAT [SEQ. ID NO:300] Oligo #248  Length: 000018 ATCGTTGTCT TCCCCATT [SEQ. ID NO:301] Oligo #249  Length: 000027 AACAACCTCA ATGGGGAAGA CGACGAT [SEQ. ID NO:302] Oligo #250  Length: 000018 ATCGTCGTCT TCCCCATT [SEQ. ID NO:303] Oligo #251  Length: 000027 AACAACCTCA ATGGTGAAGA CGAAGAT [SEQ. ID NO:304] Oligo #252  Length: 000018 ATCTTCGTCT TCCCCATT [SEQ. ID NO:305] Oligo #253  Length: 000027 AACAACCTCA ATGGTGAAGA CCACGAT [SEQ. ID NO:306] Oligo #254  Length: 000018 ATCGTGGTCT TCCCCATT [SEQ. ID NO:307] Oligo #255  Length: 000027 AACAACCTCA ATGGGGAAGA CATCGAT [SEQ. ID NO:308] Oligo #256  Length: 000018 ATCGATGTCT TCCCCATT [SEQ. ID NO:309] Oligo #257  Length: 000027 AACAACCTCA ATGGGGAAGA CTCCGAT [SEQ. ID NO:310] Oligo #258  Length: 000018 ATCGGAGTCT TCCCCATT [SEQ. ID NO:311] Oligo #259  Length: 000027 AACAACCTCA ATGGGGAAGA CCAAGCT [SEQ. ID NO:312] Oligo #260  Length: 000018 AGCTTGGTCT TCCCCATT [SEQ. ID NO:313] Oligo #261  Length: 000027 AACAACCTCA ATGGGGAAGA CCAAAAC [SEQ. ID NO:314] Oligo #262  Length: 000018 GTTTTGGTCT TCCCCATT [SEQ. ID NO:315] Oligo #263  Length: 000027 AACAACCTCA ATGGGGAAGA CCAACAG [SEQ. ID NO:316] Oligo #264  Length: 000018 CTGTTGGTCT TCCCCATT [SEQ. ID NO:317] Oligo #265  Length: 000027 AACAACCTCA ATGGGGAAGA CCAAGAA [SEQ. ID NO:318] Oligo #266  Length: 000018 TTCTTGGTCT TCCCCATT [SEQ. ID NO:319] Oligo #267  Length: 000027 AACAACCTCA ATGGGGAAGA CCAACAC [SEQ. ID NO:320] Oligo #268  Length: 000018 GTGTTGGTCT TCCCCATT [SEQ. ID NO:321] Oligo #269  Length: 000027 AACAACCTCA ATGGGGAAGA CCAAATC [SEQ. ID NO:322] Oligo #270  Length: 000018 GATTTGGTCT TCCCCATT [SEQ. ID NO:323] Oligo #271  Length: 000027 AACAACCTCA ATGGGGAAGA CCAACTG [SEQ. ID NO:324] Oligo #272  Length: 000018 CAGTTGGTCT TCCCCATT [SEQ. ID NO:325] Oligo #273  Length: 000027 AACAACCTCA ATGGGGAAGA CCAAAAA [SEQ. ID NO:326] Oligo #274  Length: 000018 TTTTTGGTCT TCCCCATT [SEQ. ID NO:327] Oligo #275  Length: 000027 AACAACCTCA ATGGGGAAGA CCAATAC [SEQ. ID NO:328] Oligo #276  Length: 000018 GTATTGGTCT TCCCCATT [SEQ. ID NO:329] Oligo #277  Length: 000027 AACAACCTCA ATGGGGAAGA CCAAGTT [SEQ. ID NO:330] Oligo #278  Length: 000018 AACTTGGTCT TCCCCATT [SEQ. ID NO:331] Oligo #279  Length: 000036 ATCGCTATGG AAAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:332] Oligo #280  Length: 000027 CCTTCGAAGG TTATTTTCCA TAGCGAT [SEQ. ID NO:333] Oligo #281  Length: 000036 ATCGAAATGG AAAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:334] Oligo #282  Length: 000027 CCTTCGAAGG TTATTTTCCA TTTCGAT [SEQ. ID NO:335] Oligo #283  Length: 000036 ATCAAAATGG AAAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:336] Oligo #284  Length: 000027 CCTTCGAAGG TTATTTTCCA TTTTGAT [SEQ. ID NO:337] Oligo #285  Length: 000036 ATCATGATGG AAAATAACCT TCGAAGGCCA AACCTG (SEQ. ID NO:338] Oligo #286  Length: 000027 CCTTCGAAGG TTATTTTCCA TCATGAT [SEQ. ID NO:339] Oligo #287  Length: 000036 ATCACCATGG AAAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:340] Oligo #288  Length: 000027 CCTTCGAAGG TTATTTTCCA TGGTGAT [SEQ. ID NO:341] O1igo #289  Length: 000036 ATCGTTATGG AAAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:342] Oligo #290  Length: 000027 CCTTCGAAGG TTATTTTCCA TAACGAT [SEQ. ID NO:343] Oligo #291  Length: 000036 ATCCTGATGC ACAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:344] Oligo #292  Length: 000027 CCTTCGAAGG TTATTGTGCA TCAGGAT [SEQ. ID NO:345] Oligo #293  Length: 000036 ATCCTGATGA TGAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:346] Oligo #294  Length: 000027 CCTTCGAAGG TTATTCATCA TCAGGAT [SEQ. ID NO:347] Oligo #295  Length: 000036 ATCCTGATGT TCAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:348] Oligo #296  Length: 000027 CCTTCGAAGG TTATTGAACA TCAGGAT [SEQ. ID NO:349] Oligo #297  Length: 000036 ATCCTGATGG CTAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:350] Oligo #298  Length: 000027 CCTTCGAAGG TTATTAGCCA TCAGGAT [SEQ. ID NO:351] Oligo #299  Length: 000036 ATCCTGATGA ACAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:352] Oligo #300  Length: 000027 CCTTCGAAGG TTATTGTTCA TCAGGAT [SEQ. ID NO:353] Oligo #301  Length: 000036 ATCCTGATGA TCAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:354] Oligo #302  Length: 000027 CCTTCGAAGG TTATTGATCA TCAGGAT [SEQ. ID NO:355] Oligo #303  Length: 000036 ATCCTGATGA AAAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:356] Oligo #304  Length: 000027 CCTTCGAAGG TTATTTTTCA TCAGGAT [SEQ. ID NO:357] Oligo #305  Length: 000036 ATCCTGATGT CCAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:358] Oligo #306  Length: 000027 CCTTCGAAGG TTATTGGACA TCAGGAT [SEQ. ID No:359] Oligo #307  Length: 000036 ATCCTGATGG TTAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:360] Oligo #308  Length: 000027 CCTTCGAAGG TTATTAACCA TCAGGAT [SEQ. ID NO:361] Oligo #309  Length: 000036 ATCCTGATGG AAAATAACCT TGCTAGGCCA AACCTG [SEQ. ID NO:362] Oligo #310  Length: 000027 CCTAGCAAGG TTATTTTCCA TCAGGAT [SEQ. ID NO:363] Oligo #311  Length: 000036 ATCCTGATGG AAAATAACCT TAACAGGCCA AACCTG [SEQ. ID NO:364] Oligo #312  Length: 000027 CCTGTTAAGG TTATTTTCCA TCAGGAT [SEQ. ID NO:365] Oligo #313  Length: 000036 ATCCTGATGG AAAATAACCT TCACAGGCCA AACCTG [SEQ. ID NO:366] Oligo #314  Length: 000027 CCTGTGAAGG TTATTTTCCA TCAGGAT [SEQ. ID NO:367] Oligo #315  Length: 000036 ATCCTGATGG AAAATAACCT TAAAAGGCCA AACCTG [SEQ. ID NO:368] Oligo #316  Length: 000027 CCTTTTAAGG TTATTTTCCA TCAGGAT [SEQ. ID NO:369] Oligo #317  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGGCT AACCTG [SEQ. ID NO:370] Oligo #318  Length: 000024 CCTGTTGAAT GCCTCCAGGT TAGC [SEQ. ID NO:371] Oligo #319  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGCGT AACCTG [SEQ. ID NO:372] Oligo #320  Length: 000024 CCTGTTGAAT GCCTCCAGGT TACG [SEQ. ID NO:373] Oligo #321  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGAAC AACCTG [SEQ. ID NO:374] Oligo #322  Length: 000024 CCTGTTGAAT GCCTCCAGGT TGTT [SEQ. ID NO:375] Oligo #323  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGGAA AACCTG [SEQ. ID NO:376] Oligo #324  Length: 000024 CCTGTTGAAT GCCTCCAGGT TTTC [SEQ. ID NO:377] Oligo #325  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGCAC AACCTG [SEQ. ID NO:378] Oligo #326  Length: 000024 CCTGTTGAAT GCCTCCAGGT TGTG [SEQ. ID NO:379] Oligo #327  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGCTG AACCTG [SEQ. ID NO:380] Oligo #328  Length: 000024 CCTGTTGAAT GCCTCCAGGT TCAG [SEQ. ID NO:381] Oligo #329  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGTTC AACCTG [SEQ. ID NO:382] Oligo #330  Length: 000024 CCTGTTGAAT GCCTCCAGGT TGAA [SEQ. ID NO:383] Oligo #331  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGACC AACCTG [SEQ. ID NO:384] Oligo #332  Length: 000024 CCTGTTGAAT GCCTCCAGGT TGGT [SEQ. ID NO:385] Oligo #333  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGTAC AACCTG [SEQ. ID NO:386] Oligo #334  Length: 000024 CCTGTTGAAT GCCTCCAGGT TGTA [SEQ. ID NO:387] Oligo #335  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGGTT AACCTG [SEQ. ID NO:388] Oligo #336  Length: 000024 CCTGTTGAAT GCCTCCAGGT TAAC [SEQ. ID NO:389] Oligo #337  Length: 000018 AAAAATCTCG CTCCATGT [SEQ. ID NO:390] Oligo #338  Length: 000016 AGCGAGATTT TTAAGAAT [SEQ. ID NO:391] Oligo #339  Length: 000018 AAAAATCTCA ACCCATGT [SEQ. ID NO:392] Oligo #340  Length: 000018 GTTGAGATTT TTAAGAAT [SEQ. ID NO:393] Oligo #341  Length: 000018 AAAAATCTCG AACCATGT [SEQ. ID NO:394] Oligo #342  Length: 000018 TTCGAGATTT TTAAGAAT [SEQ. ID NO:395] Oligo #343  Length: 000018 AAAAATCTCC ACCCATGT [SEQ. ID NO:396] Oligo #344  Length: 000018 GTGGAGATTT TTAAGAAT [SEQ. ID NO:397] Oligo #345  Length: 000018 AAAAATCTCA TCCCATGT [SEQ. ID NO:398] Oligo #346  Length: 000018 GATGAGATTT TTAAGAAT [SEQ. ID NO:399] Oligo #347  Length: 000018 AAAAATCTCA TGCCATGT [SEQ. ID NO:400] Oligo #348  Length: 000018 CATGAGATTT TTAAGAAT [SEQ. ID NO:401] Oligo #349  Length: 000018 AAAAATCTCT TCCCATGT [SEQ. ID NO:402] Oligo #350  Length: 000018 GAAGAGATTT TTAAGAAT [SEQ. ID NO:403] Oligo #351  Length: 000018 AAAAATCTCT CCCCATGT [SEQ. ID NO:404] Oligo #352  Length: 000018 GGAGAGATTT TTAAGAAT [SEQ. ID NO:405] Oligo #353  Length: 000018 AAAAATCTCA CCCCATGT [SEQ. ID NO:406] Oligo #354  Length: 000018 GGTGAGATTT TTAAGAAT [SEQ. ID NO:407] Oligo #355  Length: 000018 AAAAATCTCT ACCCATGT [SEQ. ID NO:408] Oligo #356  Length: 000018 GTAGAGATTT TTAAGAAT [SEQ. ID NO:409] Oligo #357  Length: 000027 CTGCCCCTGG CCACGGCCGC AGCTACG [SEQ. ID NO:410] Oligo #358  Length: 000024 ATGGATTGGA TGTCGCGTAG CTGC [SEQ. ID NO:411] Oligo #359  Length: 000027 CTGCCCCTGG CCACGGCCGC AGGTACG [SEQ. ID NO:412] Oligo #360  Length: 000024 ATGGATTGGA TGTCGCGTAC CTGC [SEQ. ID NO:413] Oligo #361  Length: 000027 CTGCCCCTGG CCACGGCCGC AATCACG [SEQ. ID NO:414] Oligo #362  Length: 000024 ATGGATTGGA TGTCGCGTGA TTGC [SEQ. ID NO:415] Oligo #363  Length: 000021 GCTCATCCAA TCCATATCAA G [SEQ. ID NO:416] Oligo #364  Length: 000024 ATGGATTGGA TGAGCCGTGG GTGC [SEQ. ID NO:417] Oligo #365  Length: 000021 CAGCATCCAA TCCATATCAA G [SEQ. ID NO:418] Oligo #366  Length: 000024 ATGGATTGGA TGCTGCGTGG GTGC [SEQ. ID NO:419] Oligo #367  Length: 000021 CACCATCCAA TCCATATCAA G [SEQ. ID NO:420] Oligo #368  Length: 000024 ATGGATTGGA TGGTGCGTGG GTGC [SEQ. ID NO:421] Oligo #369  Length: 000021 AAACATCCAA TCCATATCAA G [SEQ. ID NO:422] Oligo #370  Length: 000024 ATGGATTGGA TGTTTCGTGG GTGC [SEQ. ID NO:423] Oligo #371  Length: 000021 CGAGCTCCAA TCCATATCAA G [SEQ. ID NO:424] Oligo #372  Length: 000024 ATGGATTGGA GCTCGCGTGG GTGC [SEQ. ID NO:425] Oligo #373  Length: 000021 CGAAACCCAA TCCATATCAA G [SEQ. ID NO:426] Oligo #374  Length: 000024 ATGGATTGGG TTTCGCGTGG GTGC [SEQ. ID NO:427] Oligo #375  Length: 000021 CGAGACCCAA TCCATATCAA G [SEQ. ID NO:428] Oligo #376  Length: 000024 ATGGATTGGG TCTCGCGTGG GTGC [SEQ. ID NO:429] Oligo #377  Length: 000021 CGAATCCCAA TCCATATCAA G [SEQ. ID NO:430] Oligo #378  Length: 000024 ATGGATTGGG ATTCGCGTGG GTGC [SEQ. ID NO:431] Oligo #379  Length: 000021 CGAAAACCAA TCCATATCAA G [SEQ. ID NO:432] Oligo #380  Length: 000024 ATGGATTGGT TTTCGCGTGG GTGC [SEQ. ID NO:433] Oligo #381  Length: 000021 CGAATGCCAA TCCATATCAA G [SEQ. ID NO:434] Oligo #382  Length: 000024 ATGGATTGGC ATTCGCGTGG GTGC [SEQ. ID NO:435] Oligo #383  Length: 000021 CGATTCCCAA TCCATATCAA G [SEQ. ID NO:436] Oligo #384  Length: 000024 ATGGATTGGG AATCGCGTGG GTGC [SEQ. ID NO:437] Oligo #385  Length: 000021 CGATCCCCAA TCCATATCAA G [SEQ. ID NO:438] Oligo #386  Length: 000024 ATGGATTGGG GATCGCGTGG GTGC [SEQ. ID NO:439] Oligo #387  Length: 000021 CGATGGCCAA TCCATATCAA G [SEQ. ID NO:440] Oligo #388  Length: 000024 ATGGATTGGC CATCGCGTGG GTGC [SEQ. ID NO:441] Oligo #389  Length: 000021 CGATACCCAA TCCATATCAA G [SEQ. ID NO:442] Oligo #390  Length: 000024 ATGGATTGGG TATCGCGTGG GTGC [SEQ. ID NO:443] Oligo #391  Length: 000034 CATCCAATCC AAATCAAGGA CGGTGACTGG AATG [SEQ. ID NO:444] Oligo #392  Length: 000044 AATTCATTCC AGTCACCGTC CTTGATTTGG ATTGGATGTC GCGT [SEQ. ID NO:445] Oligo #393  Length: 000034 CATCCAATCG AAATCAAGGA CGGTGACTGG AATG [SEQ. ID NO:446] Oligo #394  Length: 000044 AATTCATTCC AGTCACCGTC CTTGATTTCG ATTGGATGTC GCGT [SEQ. ID NO:447] Oligo #395  Length: 000034 CATCCAATCA TGATCAAGGA CGGTGACTGG AATG [SEQ. ID NO:448] Oligo #396  Length: 000044 AATTCATTCC AGTCACCGTC CTTGATCATG ATTGGATGTC GCGT [SEQ. ID NO:449] Oligo #397  Length: 000034 CATCCAATCT TCATCAAGGA CGGTGACTGG AATG [SEQ. ID NO:450] Oligo #398  Length: 000044 AATTCATTCC AGTCACCGTC CTTGATGAAG ATTGGATGTC GCGT [SEQ. ID No:451] Oligo #399  Length: 000034 CATCCAATCT CCATCAAGGA CGGTGACTGG AATG [SEQ. ID NO:452] Oligo #400  Length: 000044 AATTCATTCC AGTCACCGTC CTTGATGGAG ATTGGATGTC GCGT [SEQ. ID NO:453] Oligo #401  Length: 000034 CATCCAATCg taATCAAGGA CGGTGACTGG AATG [SEQ. ID NO:454] Oligo #402  Length: 000044 AATTCATTCC AGTCACCGTC CTTGATTACG ATTGGATGTC GCGT [SEQ. ID NO:455] Oligo #403  Length: 000021 CGACATCCAA TCCGTATCAA G [SEQ. ID NO:456] Oligo #404  Length: 00024 ACGGATTGGA TGTCGCGTGG GTGC [SEQ. ID NO:457] Oligo #405  Length: 000021 CGACATCCAA TCAAAATCAA G [SEQ. ID NO:458] Oligo #406  Length: 000024 TTTGATTGGA TGTCGCGTGG GTGC [SEQ. ID NO:459] Oligo #407  Length: 000021 CGACATCCAA TCTACATCAA G [SEQ. ID NO:460] Oligo #408  Length: 000024 GTAGATTGGA TGTCGCGTGG GTGC [SEQ. ID NO:461] Oligo #409  Length: 000016 GCTGGTGACT GGAATG [SEQ. ID NO:462] Oligo #410  Length: 000026 AATTCATTCC AGTCACCAGC CTTGAT [SEQ. ID NO.463] Oligo #411  Length: 000016 AACGGTGACT GGAATG [SEQ. ID NO:464] Oligo #412  Length: 000026 AATTCATTCC AGTCACCGTT CTTGAT [SEQ. ID NO:465] Oligo #413  Length: 000016 GAAGGTGACT GGAATG [SEQ. ID NO:466] Oligo #414  Length: 000026 AATTCATTCC AGTCACCTTC CTTGAT [SEQ. ID NO:467] Oligo #415  Length: 000016 GGTGGTGACT GGAATG [SEQ. ID NO:468] Oligo #416  Length: 000026 AATTCATTCC AGTCACCACC CTTGAT [SEQ. ID NO:469] Oligo #417  Length: 000016 ATCGGTGACT GGAATG [SEQ. ID NO:470] Oligo #418  Length: 000026 AATTCATTCC AGTCACCGAT CTTGAT [SEQ. ID NO:471] Oligo #419  Length: 000016 CTGGGTGACT GGAATG [SEQ. ID NO:472] Oligo #420  Length: 000026 AATTCATTCC AGTCACCCAG CTTGAT [SEQ. ID NO:473] Oligo #421  Length: 000016 TTCGGTGACT GGAATG [SEQ. ID NO:474] Oligo #422  Length: 000026 AATTCATTCC AGTCACCGAA CTTGAT [SEQ. ID NO:475] Oligo #423  Length: 000016 TCCGGTGACT GGAATG [SEQ. ID NO:476] Oligo #424  Length: 000026 AATTCATTCC AGTCACCGGA CTTGAT [SEQ. ID NO:477] Oligo #425  Length: 000032 AATTCGCTAG GAAACTGACG TTCTATCTGA AA [SEQ. ID NO:478] Oligo #426  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTAGCG [SEQ. ID NO:479] Oligo #427  Length: 000032 AATTCCAGAG GAAACTGACG TTCTATCTGA AA [SEQ. ID NO:480] Oligo #428  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCTGG [SEQ. ID NO:481] Oligo #429  Length: 000032 AATTCCACAG GAAACTGACG TTCTATCTGA AA [SEQ. ID NO:482] Oligo #430  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGTGG [SEQ. ID NO:483] Oligo #431  Length: 000032 AATTCTCCAG GAAACTGACG TTCTATCTGA AA [SEQ. ID NO:484] Oligo #432  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGGAG [SEQ. ID NO:485] Oligo #433  Length: 000032 AATTCCGGAG GCGTCTGACG TTCTATCTGA AA [SEQ. ID NO:486] Oligo #434  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGACG CCTCCGG [SEQ. ID NO:487] Oligo #435  Length: 000032 AATTCCGGAG GGAACTGACG TTCTATCTGA AA [SEQ. ID NO:488] Oligo #436  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGTTC CCTCCGG [SEQ. ID NO:489] Oligo #437  Length: 000032 AATTCCGGAG GCACCTGACG TTCTATCTGA AA [SEQ. ID NO:490] Oligo #438  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGGTG CCTCCGG [SEQ. ID NO:491] Oligo #439  Length: 000032 AATTCCGGAG GATCCTGACG TTCTATCTGA AA [SEQ. ID NO:492] Oligo #440  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGGAT CCTCCGG [SEQ. ID NO:493] Oligo #441  Length: 000032 AATTCCGGAG GTCCCTGACG TTCTATCTGA AA [SEQ. ID NO:494] Oligo #442  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGGGA CCTCCGG [SEQ. ID NO:495] Oligo #443  Length: 000032 AATTCCGGAG GAAACTGACG GACTATCTGA AA [SEQ. ID NO:496] Oligo #444  Length: 000037 CTCAAGGGTT TTCAGATAGT CCGTCAGTTT CCTCCGG [SEQ. ID NO:497] Oligo #445  Length: 000032 AATTCCGGAG GAAACTGACG ATCTATCTGA AA [SEQ. ID NO:498] Oligo #446  Length: 000037 CTCAAGGGTT TTCAGATAGA TCGTCAGTTT CCTCCGG [SEQ. ID NO:499] Oligo #447  Length: 000032 AATTCCGGAG GAAACTGACG CTGTATCTGA AA [SEQ. ID NO:500] Oligo #448  Length: 000037 CTCAAGGGTT TTCAGATACA GCGTCAGTTT CCTCCGG [SEQ. ID NO:501] Oligo #449  Length: 000032 AATTCCGGAG GAAACTGACG AAATATCTGA AA [SEQ. ID NO:502] Oligo #450  Length: 000037 CTCAAGGGTT TTCAGATATT TCGTCAGTTT CCTCCGG [SEQ. ID NO:503] Oligo #451  Length: 000032 AATTCCGGAG GAAACTGACG GTTTATCTGA AA [SEQ. ID NO:504] Oligo #452  Length: 000037 CTCAAGGGTT TTCAGATAAA CCGTCAGTTT CCTCCGG [SEQ. ID NO:505] Oligo #453  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGG CT [SEQ. ID NO:506] Oligo #454  Length: 000037 CTCAAGGGTA GCCAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:507] Oligo #455  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGC GT [SEQ. ID NO:508] Oligo #456  Length: 000037 CTCAAGGGTA CGCAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:509] Oligo #457  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGA AC [SEQ. ID NO:510] Oligo #458  Length: 000037 CTCAAGGGTG TTCAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:511] Oligo #459  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGC AG [SEQ. ID NO:512] Oligo #460  Length: 000037 CTCAAGGGTC TGCAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:513] Oligo #461  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGC AC [SEQ. ID NO:514] Oligo #462  Length: 000037 CTCAAGGGTG TGCAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:515] Oligo #463  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGA TG [SEQ. ID NO:516] Oligo #464  Length: 000037 CTCAAGGGTC ATCAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:517] Oligo #465  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGT TC [SEQ. ID NO:518] Oligo #466  Length: 000037 CTCAAGGGTG AACAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:519] Oligo #467  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGT AC [SEQ. ID NO:520] Oligo #468  Length: 000037 CTCAAGGGTG TACAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:521] Oligo #469  Length: 000040 CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA [SEQ. ID NO:522] Oligo #470  Length: 000036 CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC [SEQ. ID NO:523] Oligo #471  Length: 000027 AACAACCTCA ATGGGGAAGA CCAAGAT [SEQ. ID NO:524] Oligo #472  Length: 000045 CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC [SEQ. ID NO:525] Oligo #473  Length: 000036 GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG [SEQ. ID NO:526] Oligo #474  Length: 000018 ATCTTGGTCT TCCCCATT [SEQ. ID NO:527] Oligo #475  Length: 000036 ATCCTGATGG AAAATAACCT TCGAAGGCCA AACCTG [SEQ. ID NO:528] Oligo #476  Length: 000024 GAGGCATTCA ACAGGGCTGT CAAG [SEQ. ID NO:529] Oligo #477  Length: 000015 AGTTTACAGA ATGCA [SEQ. ID NO:530] Oligo #478  Length: 000027 CCTTCGAAGG TTATTTTCCA TCAGGAT [SEQ. ID NO:531] Oligo #479  Length: 000024 CCTGTTGAAT GCCTCCAGGT TTGG [SEQ. ID NO:532] Oligo #480  Length: 000020 TTCTGTAAAC TCTTGACAGC [SEQ. ID NO:533] Oligo #481  Length: 000021 TCAGCAATTG AGAGCATTCT T [SEQ. ID NO:534] Oligo #482  Length: 000018 AAAAATCTCC TGCCATGT [SEQ. ID NO:535] Oligo #483  Length: 000048 CTGCCCCTGG CCACGGCCGC ACCCACGCGA CATCCAATCC ATATCAAG [SEQ. ID NO:536] Oligo #484  Length: 000027 CTGCCCCTGG CCACGGCCGC ACCCACG [SEQ. ID NO:537] Oligo #485  Length: 000021 CGACATCCAA TCCATATCAA G [SEQ. ID NO:538] Oligo #486  Length: 000016 GACGGTGACT GGAATG [SEQ. ID NO:539] Oligo #487  Length: 000019 GCTCTCAATT GCTGATGCA [SEQ. ID NO:540] Oligo #488  Length: 000018 CAGGAGATTT TTAAGAAT [SEQ. ID NO:541] Oligo #489  Length: 000048 ATGGATTGGA TGTCGCGTGG GTGCGGCCGT GGCCAGGGGC AGACATGG [SEQ. ID NO:542] Oligo #490  Length: 000024 GGCCGTGGCC AGGGGCAGAC ATGG [SEQ. ID NO:543] 0ligo #491  Length: 000024 ATGGATTGGA TGTCGCGTGG GTGC [SEQ. ID NO:544] Oligo #492  Length: 000026 AATTCATTCC AGTCACCGTC CTTGAT [SEQ. ID NO:545] Oligo #493  Length: 000032 AATTCCGGAG GAAACTGACG TTCTATCTGA AA [SEQ. ID NO:546] Oligo #494  Length: 000032 ACCCTTGAGA ATGCGCAGGC TCAACAGTAA TA [SEQ. ID NO:547] Oligo #495  Length: 000037 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCCGG [SEQ. ID NO:548] Oligo #496  Length: 000027 AGCTTATTAC TGTTGAGCCT GCGCATT [SEQ. ID NO:549]

TABLE 3 POLYPEPTIDES PEPTIDE #1; pMON5988 (Examp1e 9); (15-125)hIL-3         Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu [SEQ ID NO:65]         15                  20                  25 Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly         30                  35                  40 Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn         45                  50                  55 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser         60                  65                  70 Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu PrO Cys Leu Pro Leu         75                  80                  85 Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly         90                  95                  100 Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr         105                 110                 115 Leu Glu Asn Ala Gln Ala Gln Gln         120                 125 PEPTIDE #A1; pMON13304 (Example 55); Met-Ala-(15-125)hIL-3 (98I, 100R): Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu [SEQ ID NO:66]         15                  20                  25 Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly         30                  35                  40 Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn         45                  50                  55 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser         60                  65                  70 Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu         75                  80                  85 Ala Thr Ala Ala Pro Thr Arg His Pro Ile Ile Ile Arg Asp Gly         90                  95                  100 Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr         105                 110                 115 Leu Glu Asn Ala Gln Ala Gln Gln         120                 125 PEPTIDE #A2; pMON133O5 Met-Ala-(15-125)hIL-3; (95R, 98I, 100R); Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile IIe Thr His Leu [SEQ ID NO:67]         15                  20                  25 Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly         30                  35                  40 Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn         45                  50                  55 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser         60                  65                  70 Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu         75                  80                  85 Ala Thr Ala Ala Pro Thr Arg Arg Pro Ile Ile Ile Arg Asp Gly         90                  95                  100 Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr         105                 110                 115 Leu Glu Asn Ala Gln Ala Gln Gln         120                 125 PEPTIDE #A3; pMON13286 Met-Ala-(15-125)hIL-3; (42D, 45M, 46S); Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu [SEQ ID NO:69]         15                  20                  25 Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Asp         30                  35                  40 Glu Asp Met Ser Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn         45                  50                  55 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser         60                  65                  70 Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu         75                  80                  85 Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly         90                  95                  100 Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr         105                 110                 115 Leu Glu Asn Ala Gln Ala Gln Gln         120                 125

Polypeptides corresponding to SEQ ID NOS. 15, 16, 17, 18 and 129 comprising (1-133) hIL-3 containing one or more amino acid substitutions can be made using the procedures described above and in the following examples by starting with the appropriate oligonuctiotides and then constructing the DNA encoding the polypeptide and expressing it in an appropriate host cell. In a similar manner polypeptides which correspond to SEQ ID NOS. 19, 20, 21, 22 and 130 and contain one or more amino acid substitutions and wherein from 1 to 14 amino acids have been sequentially deleted from the N-terminus, or from 1 to 15 amino acids have been deleted from the C-terminus or deletions of amino acids have been made from both the N-terminus and the C-terminus can also be made by following the procedures described above and in the following examples, beginning with the appropriate starting materials.

Additional details may be found in U.S. patent application Ser. No. 07/981,044 filed Nov. 24, 1992, now abandoned which is hereby incorporated by reference in its entirety.

Additional details may be found in co filed U.S. patent application Ser. No. 08/411,795 which is hereby incorporated by reference in its entirety.

All references, patents or applications cited herein are incorporated by reference in their entirety.

Further details known to those skilled in the art may be found in T. Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982) and references cited therein, incorporated herein by reference in its entirety; and in J. Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1989) and references cited therein, incorporated herein by reference in its entirety.

The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these specific examples.

Amino acids are shown herein by standard one letter or three letter abbreviations as follows:

Abbreviated Designation Amino Acid A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine

Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.

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EXAMPLE 1

Construction of pMON 5846 (FIG. 4) which Encodes [Met-(1-133) hIL-3 (Arg129)]

A plasmid containing the gene for the cDNA of hIL-3 cloned into pUC18 on an EcoRI to HindIII fragment was obtained from British Biotechnology Limited (Cambridge, England). This plasmid was designated pPO518. The purified plasmid DNA was cleaved by the restriction endonucleases NheI and BamHI. Approximately 0.5 micrograms of cleaved plasmid DNA was ligated to 1.0 picomoles of a pair of annealed oligonucleotides with the following sequence:

5′-CTAGCGATCTTTTAATAAGCTTG-3′ [SEQ ID NO: 1]

3′-GCTAGAAAATTATTCGAACCTAG-5′ [SEQ ID NO: 2]

The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and subjected to restriction enzyme analysis. An isolate was identified in which the above oligonucleotide sequence had replaced the portion of the gene that encodes the extreme C-terminus. Within the new sequence was a new stop codon, TAA, and a recognition site for the enzyme HindIII. The new plasmid was designated pMON5846.

EXAMPLE 2

(a) Construction of Expression Vector Plasmid pMON2341

The plasmid pMON2341 was used to supply the particular replicon and expression elements used for construction of many of the plasmids used to produce hIL-3 and hIL-3 muteins in E. coli. These expression elements are described in the materials and methods section. pMON2341 is derived from pMON5515 (Olins et al., 1988) and from pMON2429. pMON2429 consists of the phage mp18 (Yanisch-Perron et al., 1985) with a BclI fragment carrying the chloramphenicol acetyl transferase (cat) gene from pBR328 (Covarrubias et al., 1981) inserted into the BamHI site. The cat gene in pMON2429 has been altered from that in pBR328 by site directed mutagenesis (Kunkel, 1985). The recognition sites for NcoI and EcoRI which occur in the native gene were altered so that these two restriction enzymes no longer recognize these sites. The changes did not alter the protein specified by the gene. Also, an NcoI site was introduced at the N-terminus of the coding sequence so that it overlaps the codon for initiator methionine.

The steps involved in construction of pMON2341 are listed below:

(1) The DNAs of pMON5515 and pMON2429 were treated with NcoI and HindIII. The fragments were ligated and used to transform competent E. coli to ampicillin resistance. From these colonies, some were identified that were chloramphenicol resistant. From one of these colonies, plasmid DNA was isolated in which the rat atriopeptigen gene of pMON5515 had been replaced by the NcoI to HindIII fragment containing the cat gene from pMON2429. This fragment contains the recognition sites for several restriction enzymes in the portion derived, from the multilinker region of mp18. The new plasmid was designated pMON2412.

(2) pMON2412 was treated with the enzyme ClaI which cleaves at one location in the pBR327 derived portion of the DNA. The protruding ends were rendered blunt by treatment with Klenow in the presence of nucleotide precursors. This DNA was mixed with an isolated 514 bp RsaI fragment derived from pEMBL8 (Dente et al., 1983). This RsaI fragment contains the origin of replication of phage f1. This ligation mixture was used to transform competent E. coli cells to ampicillin resistance. Among the plasmid DNAs isolated from these cells was pMON5578. This plasmid has the structure of pMON2412 with the f1 origin region inserted into the ClaI site. This in illustrated in the Figures and in Olins and Rangwala (1990).

(3) The DNA of pMON5578 was treated with restriction enzymes HindIII and MstII. The DNA was then treated with Klenow enzyme in the presence of nucleotide precursors to render the ends blunt. This treated DNA was ligated and used to transform competent E. coli to ampicillin resistance. From the ampicillin resistant colonies, one plasmid was recovered from which the portion between HindIII and MstII was absent. This deletion resulted in the removal of sequences from the plasmid which are recognized by a number of restriction endonuclease sites. The new plasmid was designated pMON5582.

(4) The DNA of pMON5582 was treated with SstII and BclI and ligated in the presence of annealed oligonucleotides with the sequences shown below.

5′-GGCAACAATTTCTACAAAACACTTGATACTGTATGAGCAT-3′-CGCCGTTGTTAAAGATGTTTTGTGAACTATGACATACTCGTAACAGTATAATTGCTTCAACAGAACAGATC-3′ [SEQ ID NO:3]

TGTCATATTAACGAAGTTGTCTTGT-5′ [SEQ ID NO:4]

This sequence encodes the essential elements of the recA promoter of E. coli including the transcription start site and the lexA repressor binding site (the operator) (Sancar et al., 1980). The plasmid recovered from the ligation mixes contained this recA promoter in place of the one In pMON5582 (and in pMON5515). The functionality of the recA promoter was illustrated by Olins and Rangwala (1990). The new plasmid was designated pMON5594.

(5) To eliminate the single EcoRI site in pMON5594, the DNA was treated with EcoRI, then with Klenow in the presence of nucleotide precursors to render the ends blunt and then the DNA was ligated. From this ligation mix a plasmid was recovered whose DNA was not cleaved with EcoRI. This plasmid was designated pMON5630.

(6) To alter the single recognition site for PstI, plasmid pMON5630 was subjected to site directed mutagenesis (Kunkel, 1985). The oligonucleotide used in this procedure has the sequence shown below.

5′-CCATTGCTGCCGGCATCGTGGTC-3′ [SEQ ID NO:5]

The result of the procedure was to construct pMON2341 which differs from pMON5630 in that the PstI site in the beta-lactamase gene was altered so that PstI no longer recognizes the site. The single nucleotide change does not alter the amino acid sequence of the beta-lactamase protein.

(b) Construction of pMON5847 (FIG. 5) which Encodes [Met-(1-133) hIL-3(Arg¹²⁹)]

Plasmid pMON2341 was used to supply the replicon, promotor, ribosome binding site, transcription terminator and antibiotic resistance marker for the plasmids used to produce hIL-3 in E. coli from cDNA derived hIL-3 genes.

Plasmid pMON2341 was treated with restriction enzymes NcoI and HindIII. The restriction fragment containing the replication origin was purified. The DNA of plasmid pMON5846 was treated with NcoI and HindIII. The restriction fragment containing the hIL-3 gene was gel purified. These purified restriction fragments were mixed and ligated. The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and analyzed using restriction enzymes. pMON5847 was identified as a plasmid with the replicon of pMON2341 and the hIL-3 gene in place of the chloramphenicol acetyl transferase gene. JM101 cells harboring this plasmid were cultured in M9 medium and treated with nalidixic acid as described above. Samples of the culture were examined for protein content. It was found that this hIL-3 mutein was produced at about 6% of total cell protein as measured on Coomassie stained polyacrylamide gels.

EXAMPLE 3

Construction of pMON5854 (FIG. 7) which Encodes [Met-(1-133) hIL-3(Arg¹²⁹)]

To increase the accumulation of hIL-3 in E. coli, the coding sequence of the amino terminal portion of the protein was altered to more closely reflect the codon bias found in E. coli genes that produce high levels of proteins (Gouy and Gautier, 1982). To change the coding sequence for the amino terminal portion of the gene, a pair of synthetic oligonucleotides were inserted between the NcoI and HpaI sites within the coding sequence. About 0.5 micrograms of DNA of the plasmid pMON5847 (Example 2) was treated with NcoI and HpaI. This DNA was mixed with an annealed pair of oligonucleotides with the following sequence:

5′-CATGGCTCCAATGACTCAGACTACTTCTCTTAAGACT-3′-CGAGGTTACTGAGTCTGATGAAGAGAATTCTGATCTTGGGTT-3′ [SEQ ID NO:6]

AGAACCCAA-5′ [SEQ ID NO:7]

The fragments were ligated. The ligation mixture was used to transform competent JM101 to ampicillin resistance. Colonies were picked into broth. From the cultures plasmid DNA was made and examined for the presence of a DdeI site (CTNAG) which occurs in the synthetic sequence but not between the NcoI and HpaI sites in the sequence of pMON5847. The new recombinant plasmid was designated pMON5854. The nucleotide sequence of the DNA in the coding sequence of the amino terminal portion of the hIL-3 gene in pMON5854 was determined by DNA sequencing and found to be the same as that of the synthetic oligonucleotide used in ligation. Cultures of JM101 cells harboring this plasmid were grown and treated with nalidixic acid to induce production of the hIL-3 mutant protein. Analysis of the proteins on Coomassie gels showed that the accumulation of hIL-3 mutein was about 25% of total cell protein in cultures harboring pMON5854, significantly higher than it was in cultures harboring pMON5847.

EXAMPLE 4

Construction of pMON5887 (FIG. 12) which Encodes [Met-(1-125) hIL-3]

The plasmid DNA of pMON5854 (Example 3) was treated with EcoRI and HindIII and the larger fragment was gel purified. About 0.5 mitrogram of this DNA was ligated to 1 picomole of an annealed pair of oligonucleotides which encode amino acids 107 through 125 of hIL-3. The sequences of these oligonucleotides are shown below. EcoRI to HindIII

5′-AATTCCGTCOTAAMCTGACCTTCTATCTGAAAA-3′-GGCAGCATTTGACTGGAAGATAGACTTTTCCTTGGAGAACGCGCAGGCTCAACAGTAATA-3′ [SEQ ID NO;8]

GGAACCTCTTGCGCGTCCGAGTTGTCATTATTCGA-5′ [SEQ ID NO:9]

After ligation, the DNA was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and plasmid DNA was isolated from each culture. Restriction analysis of the plasmid DNA showed the presence of an EcoRI to HindIII fragment smaller than that of pMON5854. The nucleotide sequence of the portion of the coding sequence between the EcoRI and HindIII sites was determined to confirm the accuracy of the replaced sequence. The new plasmid was designated pMON5887 encoding Met-(1-125) hIL-3 which has the following amino acid sequence:

[SEQ ID NO:10] Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln

EXAMPLE 5

Construction of pMON5967 which Encodes [Met-Ala-(15-125) hIL-3]

Plasmid DNA of pMON5887 isolated from E. coli GM48 (dam-) was cleaved with NcoI and ClaI and ligated to 1 picomole of an annealed pair of oligonucleotides, Nco I and ClaI, encoding amino acids [Met Ala (15-20) hIL-3]. The sequence of these oligonucleotides is shown below.

5′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:11]

3′-CGATTGACGAGATTGTACTAGC-5′[SEQ ID NO:12]

The resulting ligation mix was used to transform competent E. coli JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated from these cells and the size of the inserted fragment was determined to be smaller than that of pMON5887 by restriction analysis using NcoI and NsiI. The nucleotide sequence of the region between NcoI and ClaI was determined and found to be that of the synthetic oligonucleotides. The new plasmid was designated pMON5967 and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bio-assay.

EXAMPLE 6 Construction of pMON5978 which Encodes [Met-Ala-(15-125) hIL-3]

Plasmid DNA of pMON5967 isolated from E. coli GM48(dam-) was cleaved with ClaI and NsiI and ligated to 1 picomole of an annealed assembly of six oligonucleotides encoding hIL-3 amino acids 20-70 (FIG. 2). This synthetic fragment encodes three unique restriction sites, EcoRV, XhoI and PstI. The sequence of these oligonucleotides is shown in FIG. 2.

The resulting ligation mix was used to transform competent E. coli JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated and screened with XbaI and EcoRV for the presence of the new restriction site EcoRV. The DNA sequence of the region between ClaI and NsiI was determined and found to be the same as that of the synthetic oligonucleotides. The new plasmid was designated pMON5978, and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bioassay.

Plasmid pMON5978 encodes [Met-Ala-(15-125) hIL-3] which has the following amino acid sequence:

[SEQ ID NO:13] Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln

EXAMPLE 7

Construction of pMON5898

Plasmid pMON5851 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3695 base pair NcoI,HindIII fragment. The genetic elements derived from pMON5851 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and the lamB secretion leader. The AraBAD promoter is identical to that described in plasmid pMON6235 and the lamB signal peptide sequence used is that shown in FIG. 8 fused to hIL-3 at the NcoI recognition site. Plasmid pMON5873 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 408 base pair NcoI,HindIII fragment. The genetic element derived from pMON5873 is the hIL-3 gene (1-133). Clones containing the hIL-3 (1-133) gene contained a 408 base pair NcoI, HindIII restriction fragment. This construct was designated pMON5898.

EXAMPLE 8

Construction of pMON5987

Plasmid pMON6458 DNA was digested with restriction enzymes NcoI and HindIII, resulting in a 3940 base pair NcoI, HindIII fragment. The genetic elements derived from pMON6458 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and lamB secretion leader. Plasmid pMON5978 DNA was digested with NcoI and NsiI. The resulting 170 base pair NcoI, NsiI fragment encodes amino acids 15-71 of (15-125) hIL-3. Plasmid pMON5976 DNA was digested with NsiI and HindIII. The resulting 175 base pair NsiI, HindIII fragment encodes amino acids 72-125 of (15-125) hIL-3. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and screened for the restriction sites EcoRV and NheI and DNA sequenced to confirm the correct insert.

EXAMPLE 9

Construction of pMON5988

The plasmid DNA of pMON5987 was digested with NheI and EcoRI, resulting in a 3903 base pair NheI, EcoRI fragment. The 3903 base pair NheI, EcoRI fragment was ligated to 1.0 picomoles of the following annealed oligonucleotides (oligo #3 and Oligo #4):

5′-CTAGCCACGGCCGCACCCACGCGACATCCAATCCATATCAA-3′-GGTGCCGGCGTGGGTGCGCTGTAGGTTAGGTATAGTTGGACGGTGACTGGAATG-3′ [SEQ ID NO:131]

CCTGCCACTGACCTTACAATT-5′ [SEQ ID NO:132]

The ligation reaction mixture was used to transform E. coli K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm positive clones.

This plasmid was constructed to change alanine 101 to aspartic acid in the hIL-3 gene (15-125). The Ala¹⁰¹ to Asp¹⁰¹ change was confirmed by DNA sequencing. This plasmid was designated pMON5988 and encodes Peptide #1 [SEQ ID NO:65].

EXAMPLE 10

Construction of pMON5873 which encodes [Met-(1-133) hIL-3]

The gene obtained from British Biotechnology, Ltd. specified arginine at codon position 129. The amino acid specified in the native hIL-3 cDNA is serine. To produce a protein with the native sequence at this position, the portion of the coding sequence between the EcoRI site at codons 106 and 107 and the NheI site at codons 129 and 130 was replaced. Plasmid DNA of pMON5854 (Example 3) and pMON5853 (Example 64) were treated with EcoRI and NheI. The larger fragments of each were gel purified. These were ligated to a pair of an annealed oligonucleotides with the following sequences:

5′-AATTCCGTCGTAAACTGACCTTCTATCTGAAAACC-3′-GGCAGCATTTGACTGGAAGATAGACTTTTGGTTGGAGAACGCGCAGGCTCAACAGACCACTCTGTCG-3′ [SEQ ID NO: 136]

AACCTCTTGCGCGTCCGAGTTGTCTGGTGAGACAGCGATC-5′ [SEQ ID NO:137]

The ligation reaction mixtures were used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and grown. Plasmid DNA was isolated and screened for the presence of a new StyI recognition site present in the synthetic DNA and not in pMON5854 and pMON5853. The nucleotide sequence of the gene in the region between EcoRI and NheI was determined and found to be that of the synthetic oligonucleotides. The new plasmids were designated pMON5873 encoding [Met-(1-133) hIL-3] and pMON5872 encoding [Met-(15-133) hIL-3].

The plasmid, pMON5873, encodes Met-(1-133) hIL-3 which has the following amino acid sequence:

[SEQ ID NO:128] Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe

EXAMPLE 11

Construction of pMON6458

Plasmid pMON6525 DNA was digested with restriction enzymes HindIII and SalI and the resulting 3172 base pair fragment was isolated from a 1% agarose gel by interception onto DEAE membrane. The genetic elements derived from pMON6525 are the beta-lactamase gene (AMP), pBR327 origin of replication, and phage f1 origin of replication as the transcription terminator. (The genetic elements derived from plasmid pMON6525 are identical to those in plasmid pMON2341 which could also be used to construct pMON6458.) Plasmid pMON6457 was, digested with restriction enzymes HindIII and SalI and the resulting 1117 base pair fragment was isolated by PAGE and crush and soak elution. The genetic elements derived from pMON6457 are the pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and the (15-125) hIL-3 gene. The restriction fragments were ligated and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 15-125) contained a 345 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6458. This plasmid was constructed to eliminate an EcoRI restriction site outside the hIL-3 gene coding region in plasmid pMON6457.

EXAMPLE 12

Construction of pMON6455

Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMON5905 are the beta-lactamase gene (AMP), pBR327 origin of replication, pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and phage f1 origin of replication as the transcription terminator. The following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, g10L ribosome binding site and phage f1 origin of replication as the transcription terminator, derived from plasmid pMON5905 are identical to those in plasmid pMON5594 which could also be used to construct pMON6455. The AraBAD promoter is identical to that described in pMON6235. The lamB signal peptide sequence used in pMON6455 is that shown in FIG. 8 fused to hIL-3 (15-125) at the NcoI site. Plasmid pMON5887 DNA was digested with restriction enzymes HindIII and NcoI, resulting in a 384 base pair NcoI, HindIII fragment. The restriction fragments were ligated, and the ligation reaction mixture was used to transform into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Positive clones containing the hIL-3 gene (encoding amino acids 1-125) contained a 384 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6455.

EXAMPLE 13

Construction of pMON6456

Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMONS905 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, pAraBAD promoter, g10L ribosome binding site and the lamB secretion leader. Plasmid pMON5871 was digested with restriction enzymes HindIII and NcoI, resulting in a 330base pair NcoI, HindIII fragment. The genetic element derived from pMON5871 encompassed the bases encoding the (1-107) hIL-3 gene. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 1-107) contained a 330 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6456.

EXAMPLE 14

Construction of pMON6457

Plasmid pMON6455 DNA grown in E. coli strain GM48 (dam-)was digested with restriction enzymes NcoI and ClaI, resulting in a 4293 base pair NcoI, ClaI fragment. The restriction fragment was ligated to 1.0 picomoles of annealed oligonucleotides (Oligo #5 and Oligo #6) with the following sequence coding for Met Ala 14-20 hIL-3:

5′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:151]

3′-CGATTGACCAGATTGTACTAGC-5′[SEQ ID NO:152]

The resulting DNA was transformed into E. coli K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes XbaI and EcoRI in double digest. Positive clones containing the hIL-3 gene (encoding aa 15-125 of hIL-3) contained a 433 base pair XbaI, EcoRI restriction fragment and were DNA sequenced. This construct was designated pMON6457. This plasmid was constructed to delete the first 14 amino acids of hIL-3. The coding sequence of the resulting gene begins as follows:

5′ ATG GCT AAC TGC . . . 3′ [SEQ ID NO:153] Met Ala Asn Cys . . . [SEQ ID NO:154]          15

The first two amino acids (Methionine, Alanine) create an NcoI restriction site and a signal peptidase cleavage site between the lamB signal peptide and (15-125) hIL-3. Plasmid pMON6457 encodes (15-125) hIL-3 which has the amino acid sequence designated SEQ ID NO 65.

EXAMPLE 15

Construction of pMON6235

One of the DNA fragments used to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotides, Oligo #51(A) [SEQ ID NO:155] and Oligo #52(A) [SEQ ID NO:156], were used as primers in this procedure. The template for the PCR reaction was E. coli strain W3110 chromosomal DNA, prepared as described in Maniatis (1982). The oligonucleotide primers were designed to amplify the AraBAD promoter (Greenfield et al., 1978). The resulting DNA product was digested with the restriction enzymes SacII and BglII. The reaction mixture was purified as described previously. Plasmid, pMON5594, DNA was digested with SacII and BglII, resulting in a 4416 base pair SacII,BglII restriction fragment which contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, G10L ribosome binding site, phage f1 origin of replication as the transcription terminator and the chloramphenicol acetyl transferase (cat) gene. The 4416 base pair SacII,BglII restriction fragment from pMON5594 was ligated to the PCR-generated SacII, BglII DNA fragment. The ligation mixture was used to transform E. coli K-12 strain JM101. Positive clones contained a 323 base pair SacII,BglII fragment and were DNA sequenced to confirm that the SacII,BglII fragment was the AraBAD promoter. This construct was designated pMON6235.

EXAMPLE 16

Construction of pMON6460

One of the DNA fragments to construct this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the oligonucleotides, Oligo #7 [SEQ ID NO: 26] and Oligo #8 [SEQ ID NO: 27] as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. Upon completion, the digest was heated at 70° C. for 15 minutes to inactivate the enzymes. The restriction fragment was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH₄OAc. The oligonucleotide, Oligo #8, introduces two stop codons (TAA) after amino acid 93 of hIL-3 and Creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones containing the above mentioned changes released a 1023 base pair SalI fragment. This construct was designated pMON6460. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 94, 95, 96 and 97 of hIL-3.

EXAMPLE 17

Construction of pMON6461

One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #9 [SEQ. ID NO: 28], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #9, introduces two stop codons (TAA) after amino acid 97 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1035 base pair SalI fragment. This construct was designated pMON6461. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 98, 99, 100 and 101 of hIL-3.

EXAMPLE 18

Construction of pMON6462

One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #10 [SEQ. ID NO: 31], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #10 [SEQ. ID NO: 31] introduces two stop codons (TAA) after amino acid 101 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1047 base pair SalI fragment. This construct was designated pMON6462. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 102, 103, 104 and 105 of hIL-3.

EXAMPLE 19

Construction of Single Amino Acid Substitution Libraries at Positions 94, 95, 96 and 97

One of the DNA fragments used to construct the plasmids containing single amino acid substitution at positions 94, 95, 96 and 97 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction plasmid pMON6460 DNA was the template and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus, The degenerate oligonucleotides, Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35], were the primers at the C-terminus. These oligonucleotides are 32-fold degenerate, with G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 94, 95, 96 and 97 of hIL-3 respectively. These degenerate oligonucleotide primers theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at a single position. The degenerate oligonucleotides (Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35]) replace the twelve bases introduced into pMON6460, that encode the two stop codons (TAA) after amino acid 93 of hIL-3 and the SalI recognition sequence. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. The resulting PCR-generated DNA products were digested with the restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6460 was ligated to the PCR-generated NcoI, EcoRI DNA fragments. Plasmid DNA from individual colonies was isolated as described previously and screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #15 [SEQ. ID NO: 36], as the probe which had been labeled with P³². Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 20

Construction of Single Amino Acid Substitution Libraries at Positions 98. 99, 100 and 101

Single amino acid substitutions variants were constructed at position 98, 99, 100 and 101 as described previously, with the following changes. In the PCR reaction the template was plasmid pMON6461 DNA and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #16 [SEQ. ID NO: 37], Oligo #17 [SEQ. ID NO: 38], Oligo #18 [SEQ. ID NO: 39] and Oligo #19 [SEQ. ID NO: 40], were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6461 was ligated to the PCR-generated DNA NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #20 [SEQ. ID NO: 41], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 21

Construction of Single Amino Acid Substitution Libraries at Positions 102, 103, 104 and 105

Single amino acid substitutions variants were constructed at position 102, 103, 104 and 105 as described previously, with the following changes. The template was pMON6462 and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #21 [SEQ. ID NO: 42], Oligo #22 [SEQ. ID NO: 43], Oligo #23 [SEQ. ID NO: 44] and Oligo #24 [SEQ. ID NO: 45] were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes, NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6462 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #25 [SEQ. ID NO: 46], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 22

Construction of Plasmid pMON6464

Amino acids 17-22 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the PCR reaction using the oligonucleotides, Oligo #26 and Oligo #27 as primers. The resulting PCR-generated DNA products were purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 17-22 of hIL-3 have been deleted. pMON6464 was made to serve as the template for the creation of single amino acid substitution variants at positions 17, 18, 19, 20, 21 and 22 of hIL-3.

EXAMPLE 23

Construction of Plasmid pMON6465

Amino acids 23-28 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo # 26 and Oligo #28, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 23-28 of hIL-3 have been deleted. pMON6465 was made to serve as the template for the creation of single amino acid substitution variants at positions 23, 24, 25, 26, 27 and 28 of hIL-3.

EXAMPLE 24

Construction of Plasmid pMON6466

Amino acids 29-34 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #29 as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 29-34 of hIL-3 have been deleted. pMON6466 was made to serve as the template for the creation of single amino acid substitution variants at positions 29, 30, 31, 32, 33 and 34 of hIL-3.

EXAMPLE 25

Construction of Plasmid pMON6467

Amino acids 35-40 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #30, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRV. The NcoI, EcoRV restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, EcoRV restriction fragment. Positive clones contained a 81 base pair NcoI, EcoRV restriction fragment in which the bases encoding amino acids 35-40 of hIL-3 have been deleted. pMON6467 was made to serve as the template for the creation of single amino acid substitution variants at positions 35, 36, 37, 38, 39 and 40 of hIL-3.

EXAMPLE 26

Construction of Plasmid pMON6468

Amino acids 41-46 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #31, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 41-46 of hIL-3 have been deleted. pMON6468 was made to serve as the template for the creation of single amino acid substitution variants at positions 41, 42, 43, 44, 45 and 46 of hIL-3.

EXAMPLE 27

Construction of Plasmid pMON6469

Amino acids 47-52 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #32, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 47-52 of hIL-3 have been deleted. pMON6469 was made to serve as the template for the creation of single amino acid substitution variants at positions 47, 48, 49, 50, 51 and 52 of hIL-3.

EXAMPLE 28

Construction of Plasmid pMON6470

Amino acids 53-58 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #33, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 53-58 of hIL-3 have been deleted. pMON6470 was made to serve as the template for the creation of single amino acid substitution variants at positions 53, 54, 55, 56, 57 and 58 of hIL-3.

EXAMPLE 29

Construction of Plasmid pMON6471

Amino acids 59-64 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #34, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 59-64 of hIL-3 have been deleted. pMON6471 was made to serve as the template for the creation of single amino acid substitution variants at positions 59, 60, 61, 62, 63 and 64 of hIL-3.

EXAMPLE 30

Construction of Plasmid pMON6472

Amino acids 65-70 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #35, as primers. The resulting PCR-generated DNA product was purified and digested with EcoRI and XhoI. The EcoRI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated EcoRI, XhoI restriction fragment. Positive clones contained a 126 base pair EcoRI, XhoI restriction fragment in which the bases encoding amino acids 65-70 of hIL-2 have been deleted. pMON6472 was made to serve as the template for the creation of single amino acid substitution variants at positions 65, 66, 67, 68, 69 and 70 of hIL-3.

EXAMPLE 31

Construction of Plasmid pMON6473

Amino acids 71-76 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #36, as primers. The resulting PCR-generated DNA product was and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment, in which the bases encoding amino acids 71-76 of hIL-3 have been deleted, and lost the NsiI restriction site. pMON6473 was made to serve as the template for the creation of single amino acid substitution variants at positions 71, 72, 73, 74, 75 and 76 of hIL-3.

EXAMPLE 32

Construction of Plasmid pMON6474

Amino acids 77-82 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #37, as primers. The resulting PCR-generated DNA product was purified and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 77-82 of hIL-3 have been deleted. pMON6474 was made to serve as the template for the creation of single amino acid substitution variants at positions 77, 78, 79, 80, 81 and 82 of hIL-3.

EXAMPLE 33

Construction of Plasmid pMON6475

Amino acids 83-88 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #38, as primers. The resulting PCR-generated DNA product was digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 83-88 of hIL-3 have been deleted. pMON6475 was made to serve as the template for the creation of single amino acid substitution variants at positions 83, 84, 85, 86, 87 and 88 of hIL-3.

EXAMPLE 34

Construction of Plasmid pMON6476

Amino acids 88-93 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #39, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the-bases encoding amino acids 88-93 of hIL-3 have been deleted. pMON6476 was made to serve as the template for the creation of single amino acid substitution variants at positions 88, 89, 90, 91, 92 and 93 of hIL-3.

EXAMPLE 35

Construction of Plasmid pMON6477

Amino acids 106-111 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #40, as primers. The resulting PCR-generated DNA fragment was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 106-111 of hIL-3 have been deleted. pMON6477 was made to serve as the template for the creation of single amino acid substitution variants at positions 106, 107, 108, 109, 110 and 111 of hIL-3.

EXAMPLE 36

Construction of Plasmid pMON6478

Amino acids 112-117 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #41, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The 4008 bp NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 112-117 of hIL-3 have been deleted. pMON6478 was made to serve as the template for the creation of single amino acid substitution variants at positions 112, 113, 114, 115, 116 and 117 of hIL-3.

EXAMPLE 37

Construction of Plasmid pMON6479

Amino acids 118-123 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, oligo #7 and Oligo #42, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 118-123 of hIL-3 have been deleted. pMON6479 was made to serve as the template for the creation of single amino acid substitution variants at positions 118, 119, 120, 121, 122 and 123 of hIL-3.

EXAMPLE 38

Construction of Single Amino Acid Substitution Libraries at Positions 17, 18, 19, 20, 21 and 22

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 17, 18, 19, 20, 21 and 22 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6464 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #43, Oligo #44, Oligo #45, Oligo #46, Oligo #47 and Oligo #48 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6464. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 17, 18, 19, 20, 21 and 22 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA product was digested with NcoI and EcoRV. Plasmid pMON6464 DNA was digested with restriction enzymes NcoI and EcoRV resulting in a 4190 base pair fragment which was ligated to the PCR-generated NcoI, EcoRV restriction fragments. Plasmid DNA was isolated and screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #139, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 39

Construction of Single Amino Acid Substitution Libraries at Positions 23, 24, 25, 26, 27 and 28

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 23, 24, 25, 26, 27 and 28 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6465 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #49, Oligo #50, Oligo #51, Oligo #52, Oligo #53 and Oligo #54 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6465. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 23, 24, 25, 26, 27 and 28 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRV. Plasmid pMON6465 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #140, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 40

Construction of Single Amino Acid Substitution Libraries at Positions 29, 30, 31, 32, 33 and 34

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 29, 30, 31, 32, 33 and 34 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6466 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #55, Oligo #56, Oligo #57, Oligo #58, Oligo #59 and Oligo #60 were the primers at the C-terminus. The oligonucleotide Oligo #26 was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6466. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 29, 30, 31, 32, 33 and 34 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6466 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #141, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 41

Construction of Single Amino Acid Substitution Libraries at Positions 35, 36, 37, 38, 39 and 40

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 35, 36, 37, 38, 39 and 40 of hIL-3 were generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6467 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #61, Oligo #62, Oligo #63, Oligo #64, Oligo #65 and Oligo #66 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6467. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 35, 36, 37, 38, 39 and 40 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored and at the other position, 32 different codons substitutions were created at positions independently. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6467 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments.

Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #142, which had been labeled with p32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 42

Construction of Single Amino Acid Substitution Libraries at Positions 41, 42, 43, 44, 45 and 46

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 41, 42, 43, 44, 45 and 46 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6468 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #67, Oligo #68, Oligo #69, Oligo #70, Oligo #71 and Oligo #72 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6468. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 41, 42, 43, 44, 45 and 46 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6468 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #143, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 43

Construction of Single Amino Acid Substitution Libraries at Positions 47, 48, 49, 50, 51 and 52

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 47, 48, 49, 50, 51 and 52 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6469 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #73, Oligo #74, Oligo #75, Oligo #76, Oligo #77 and Oligo #78 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6469. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 47, 48, 49, 50, 51 and 52 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6469 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #143, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 44

Construction of Single Amino Acid Substitution Libraries at Positions 53, 54, 55, 56, 57 and 58

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 53, 54, 55, 56, 57 and 58 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6470 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #79, Oligo #80, Oligo #81, Oligo #82, Oligo #83 and Oligo #84 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6470. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 53, 54, 55, 56, 57 and 58 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6470 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #145, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 45

Construction of Single Amino Acid Substitution Libraries at Positions 59, 60, 61, 62, 63 and 64

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 59, 60, 61, 62, 63 and 64 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6471 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #85, Oligo #86, Oligo #87, Oligo #88, Oligo #89 and Oligo #90 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6471. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 59, 60, 61, 62, 63 and 64 of hIL-3 respectively, These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6471 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #146, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 46

Construction of Single Amino Acid Substitution Libraries at Positions 65, 66, 67, 68, 69 and 70

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 65, 66, 67, 68, 69 and 70 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6472 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #91, Oligo #92, Oligo #93, Oligo #94, Oligo #95 and Oligo #96 , were the primers at the N-terminus. The oligonucleotide, Oligo 26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6472. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 65, 66, 67, 68, 69 and 70 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and XhoI. Plasmid pMON6472 DNA was digested with restriction enzymes EcoRI and XhoI and the resulting 4145 base pair fragment was ligated to the PCR-generated EcoRI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #147, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 47

Construction of Single Amino Acid Substitution Libraries at Positions 71, 72, 73, 74, 75 and 76

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 71, 72, 73, 74, 75 and 76 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6473 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #97, Oligo #98, Oligo #99, Oligo #100, Oligo #101 and Oligo #102 , were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6473. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 71, 72, 73, 74, 75 and 76 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA fragments were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6473 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #148, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 48

Construction of Single Amino Acid Substitution Libraries at Positions 77, 78, 79, 80, 81 and 82

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 77, 78, 79, 80, 81 and 82 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the reaction the plasmid pMON6474 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #103, Oligo #104, Oligo #105, Oligo #106, Oligo #107 and Oligo #108, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6474. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 77, 78, 79, 80, 81 and 82 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and PstI as described previosly. Plasmid pMON6474 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #149, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 49

Construction of Single Amino Acid Substitution Libraries at Positions 83, 84, 85, 86, 87 and 88

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 83, 84, 85, 86, 87 and 88 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6475 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #109, Oligo #110, Oligo #111, Oligo #112, Oligo #113 and Oligo #114, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used an the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6475. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 83, 84, 85, 86, 87 and 88 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA producs were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6475 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #150, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 50

Construction of Single Amino Acid Substitution Libraries at Positions 88, 89, 90, 91, 92 and 93

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 88, 89, 90, 91, 92 and 93 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6476 DNA was the template and the following degenerate oligonucleotides, Oligo #114, Oligo #115, Oligo #116, Oligo #117, Oligo #118 and Oligo #119, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6476. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 88, 89, 90, 91, 92 and 93 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and NcoI. Plasmid pMON6476 DNA was digested with restriction enzymes EcoRI and NcoI and the resulting 4008 base pair fragment was ligated to the PCR-generated EcoRI, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #151, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 51

Construction of Single Amino Acid Substitution Libraries at Positions 106, 107, 108, 109, 110 and 111

One of the DNA fragments used to construct the plasmids containing the single amino acid substitutions at positions 106, 107, 108, 109, 110 and 111 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously in two sequential PCR reactions. In the first PCR reaction, plasmid pMON6477 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #120, Oligo #121, Oligo #122, Oligo #123, Oligo #124 and Oligo #125 were the primers at the C-terminus. The oligonucleotide, Oligo #7 was the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON9477. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 106, 107, 108, 109, 110 and 111 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The DNA generated in this PCR reaction was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH₄OAc to remove any primer that was not extended. This DNA was then used as a primer in the second PCR reaction.

In the second PCR reaction plasmid pMON6477 DNA was the template, the DNA product generated in the first PCR reaction (described above) was the primer at the N-terminus and the oligonucleotide, Oligo #126 (DNA sequence shown in Table 1), was the primer at the C-terminus. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6477 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #152, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 52

Construction of Single Amino Acid Substitution Libraries at Positions 112, 113, 114, 115, 116 and 117

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 112, 113, 114, 115, 116 and 117 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6478 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #127, Oligo #128, Oligo #129, Oligo #130, Oligo #131 and Oligo #132, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus, The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6478. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 112, 113, 114, 115, 116 and 117 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6478 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #153, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 53

Construction of Single Amino Acid Substitution Libraries at Positions 118, 119, 120, 121, 122 and 123

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 118, 119, 120, 121, 122 and 123 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6479 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #133, Oligo #134, Oligo #135, oligo #136, Oligo #137 and Oligo #138, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6479. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 118, 119, 120, 121, 122 and 123 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6479 DNA was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #154, which had been labeled with P³². Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 54

Construction of pMON13358

Plasmid pMON5978 DNA (Example 6) was digested with restriction enzymes NsiI and EcoRI and the resulting 3853 base pair NsiI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-71 and 106-125 of (15-125) hIL-3. The 3853 base pair NsiI,EcoRI restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

Oligo #15(A) [SEQ ID NO: 29]

Oligo #16(A) [SEQ ID NO: 30]

In the resulting plasmid the 111 bases between the NsiI and EcoRI restriction sites in the (15-125) hIL-3 gene are replaced with 24 bases from the above mentioned oligonucleotides. This linker also creates a NdeI recognition sequence.

EXAMPLE 55

Construction of pMON13304

Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

Oligo #155 [SEQ ID NO:200] Oligo #156 [SEQ ID NO:201] Oligo #157 [SEQ ID NO:202] Oligo #158 [SEQ ID NO:203] Oligo #159 [SEQ ID NO:204] Oligo #160 [SEQ ID NO:205] Oligo #161 [SEQ ID NO:206] Oligo #162 [SEQ ID NO:207]

When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13304, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A1 [SEQ ID NO:66]

EXAMPLE 56

Construction of pMON13305

Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI, EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

Oligo #155 [SEQ ID NO:200] Oligo #156 [SEQ ID NO:201] Oligo #157 [SEQ ID NO:202] Oligo #158 [SEQ ID NO:203] Oligo #159 [SEQ ID NO:204] Oligo #160 [SEQ ID NO:205] Oligo #163 [SEQ ID NO:208] Oligo #164 [SEQ ID NO:209]

When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 95R, 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13305, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A2 [SEQ ID NO:67]

EXAMPLE 57

Construction of pMON13286

Plasmid pMON5978 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 3865 base pair NcoI,EcoRV fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, precA promoter, g10L ribosome binding site and the bases encoding amino acids 47-125 of (1-125) hIL-3. The 3865 base pair NcoI,EcoRV restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

Oligo #165 [SEQ ID NO:210] Oligo #166 [SEQ ID NO:211] Oligo #167 [SEQ ID NO: 212] Oligo #168 [SEQ ID NO:213] Oligo #169 [SEQ ID NO:214] Oligo #170 [SEQ ID NO:215]

When assembled, the oligonucleotides create NcoI and EcoRV restriction ends and the DNA sequence that encodes amino acids 15-46 of (15-125) hIL-3 with the following amino acid substitutions; 42D, 45M and 46S. The codons encoding amino acids 15-46 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13286, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A3 SEQ ID NO:69 DNA sequence #A4 pMON13286 42D, 45M, 46S ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGAC GAAGACATGT CTATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA AGTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG SEQ ID NO:68

EXAMPLE 58

Construction of pMON5853 (FIG. 6) which Encodes [Met-(15-133) hIL-3 (Arg129)]

Plasmid DNA of pMON5847 (Example 2) was treated with NcoI. The restriction enzyme was inactivated by heat treatment (65° C. for 10 minutes). The DNA was then treated with large fragment of DNA polymerase I (Klenow) in the presence of all four nucleotide precursors. This produces DNA termini with non-overlapping ends. After 5 minutes at 37° C., the polymerase was inactivated by heat treatment at 65° C. for 10 minutes. The DNA was then treated with HpaI, an enzyme which produces non-overlapping termini. The DNA was ethanol precipitated and ligated. The ligation reaction mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked and plasmid DNA was analyzed by restriction analysis. A plasmid designated pMON5853 was identified as one containing a deletion of the amino terminal 14 codons of the hIL-3 gene. The DNA sequence for the junction of the ribosome binding site to the (15-133) hIL-3 gene was determined to be the following:

5′-AAGGAGATATATCCATGAACTGCTCTAAC-3′ [SEQ ID NO:133]                  M  N  C  S  N [SEQ ID NO:134]

The lower line contains the one-letter code for the amino acids specified by the coding sequence of the amino terminus of the 15-133 hIL-3 gene. These are methionine, asparagine, cysteine, serine and asparagine.

When cultures of JM101 cells harboring this plasmid were induced with nalidixic acid, it was found that hIL-3 (15-133) accumulated at levels higher than hIL-3 (pMON5847).

The plasmid, pMON5853, encodes Met-(15-133) hIL-3 (Arg¹²⁹) which has the following amino acid sequence:

    Met Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Arg Leu Ala Ile Phe [SEQ ID NO:135]

Formula XI shown below is a representation of a [(15-125) hIL-3 mutein] with numbers in bold type added above the amino acids to represent the position at which the amino acid below the bolded number appears in native (1-133) hIL-3 [e.g. the amino acid at position 1 of Formula XI corresponds to the Asn which appears at position 15 in native (1-133) hIL-3]. The number shown in bold indicates the amino acids that correspond to the native IL-3(1-133). The non-bold members below the amino acids sequences are for Seq Id reference numbers. When the muteins are expressed the initial amino acid may be preceded by Met- or Met-Ala-.

 15                  20                  25 Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln [SEQ ID NO:23]  1                5                  10                  15  30                  35                  40 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp                  20                  25                  30  45                  50                  55 Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu                  35                  40                  45  60                  65                  70 Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile                  50                  55                  60  75                  80                  85 Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr                  65                  70                  75  90                  95                 100 Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp                  80                  85                  90 105                 110                 115 Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu                  95                 100                 105 120                 125 Asn Ala Gln Ala Gln Gln                 110

Table 6 shows (15-125) hIL-3 muteins of the present invention which have one (and in some cases two) amino acid substitutions in the (15-125) hIL-3 polypeptide and which were constructed as described in the Examples. The mutants in Table 6 were secreted into the periplasmic space in E. coli. The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (15-125) hIL-3 (pMON6458 or pMMON5988). The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “-” indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

TABLE 6 (15-125) HUMAN INTERLEUKIN-3 MUTANTS hIL-3 aa PARENTAL (15-125)hIL-3 MUTANT POSITION¹ aa CODON aa SEQ ID NO: CODON BIOL ACTIVITY 17/3¹ SER TCT LYS 19 AAG <0.018 (1) 17/3 SER TCT GLY 19 GGG 1.2 ± 1.1 (3) 17/3 SER TCT ASP 19 GAC 1.0 ± 0.7 (3) 17/3 SER TCT MET 19 ATG 0.50 (1) 17/3 SER TCT GLN 19 CAG 1.2 ± 0.7 (3) 17/3 SER TCT ARG 19 AGG <0.070 (1) 18/4 ASN AAC HIS 19 CAC 1.2 ± 0.3 (3) 18/4 ASN AAC LEU 19 CTC 0.45 ± 0.42 (4) 18/4 ASN AAC ILE 19 ATC 1.5 ± 0.2 (2) 18/4 ASN AAC PHE 19 TTC 0.19 ± 0.26 (2) 18/4 ASN AAC ARG 19 CGG 0.10 (1) 18/4 ASN AAC GLN 19 CAA 0.37 (1) 19/5 MET ATG PHE 19 TTC 0.25 (1) 19/5 MET ATG ILE 19 ATG 0.77 ± 0.70 (9) 19/5 MET ATG ARG 19 AGG 0.17 (1) 19/5 MET ATG GLY 19 GGA 0.06 (1) 19/5 MET ATG ALA 19 GCG 0.19 (1) 19/5 MET ATG CYS 19 TGC — 20/6 ILE ATC CYS 19 TGC — 20/6 ILE ATC GLN 19 CAG — 20/6 ILE ATC GLU 19 GAG <0.025 (1) 20/6 ILE ATC ARG 19 CGC <0.025 (1) 20/6 ILE ATC PRO 19 CCG 0.29 ± 0.16 (3) 20/6 ILE ATC ALA 19 GCG 0.18 (1) 21/7 ASP GAT PHE 19 TTC <0.016 (1) 21/7 ASP GAT LYS 19 AAG 0.027 ± 0.027 (2) 21/7 ASP GAT ARG 19 AGG <0.008 (1) 21/7 ASP GAT ALA 19 GCG 0.07 ± 0.06 (3) 21/7 ASP GAT GLY 19 GGG 0.032 (1) 21/7 ASP GAT VAL 19 GTG <0.008 (1) 22/8 GLU GAA TRP 19 TGG — 22/8 GLU GAA PRO 19 CCG <0.015 (1) 22/8 GLU GAA SER 19 TCG <0.015 (1) 22/8 GLU GAA ALA 19 GCC <0.015 (1) 22/8 GLU GAA HIS 19 CAC <0.015 (1) 22/8 GLU GAA GLY 19 GGC <0.008 (1) 23/9 ILE ATT VAL 19 GTG 0.18 (1) 23/9 ILE ATT ALA² 19 GCG 1.16 ± 0.16 (3) 23/9 ILE ATT LEU 19 TTG 1.3 (1) 23/9 ILE ATT GLY² 19 GGG 0.06 (1) 23/9 ILE ATT TRP 19 TGG — 23/9 ILE ATT LYS² 19 AAG — 23/9 ILE ATT PHE 19 TTC — 23/9 ILE ATT LEU² 19 TTG 3.0 ± 1.1 (3) 23/9 ILE ATT SER² 19 AGC <0.005 (1) 23/9 ILE ATT ARG² 19 CGC — 24/10 ILE ATA GLY 19 GGG <0.004 (1) 24/10 ILE ATA VAL 19 GTC 0.89 ± 0.23 (4) 24/10 ILE ATA ARG³ 19 CGG — 24/10 ILE ATA SER 19 AGC <0.003 (1) 24/10 ILE ATA PHE 19 TTC 0.29 ± 0.24 (2) 24/10 ILE ATA LEU 19 CTG 0.52 ± 0.12 (3) 25/11 THR ACA HIS 19 CAC 1.11 ± 0.2 (3) 25/11 THR ACA GLY 19 GGC 0.48 ± 0.27 (4) 25/11 THR ACA GLN 19 CAG 1.0 ± 0.8 (4) 25/11 THR ACA ARG 19 CGG 0.26 ± 0.17 (2) 25/11 THR ACA PRO 19 CCG 0.36 (1) 31/17 PRO CCT GLY 19 GGG 0.79 ± 0.61 (2) 31/17 PRO CCT ALA 19 GCC 0.49 (1) 31/17 PRO CCT ARG 19 CGC 0.25 ± 0.20 (2) 31/17 PRO CCT LEU 19 CTG 0.22 (1) 31/17 PRO CCT GLN 19 CAG 0.62 ± 0.04 (2) 31/17 PRO CCT LEU⁴ 19 CTG 0.30 ± 0.20 (3) 32/18 LEU TTG VAL² 19 GTG 0.01 (1) 32/18 LEU TTG ARG 19 CCC 1.5 ± 1.0 (4) 32/18 LEU TTG CLN 19 CAG 0.93 ± 0.18 (3) 32/18 LEU TTG ASN 19 AAC 1.2 ± 0.5 (5) 32/18 LEU TTG GLY⁵ 19 GGC 0.84 ± 1.0 (3) 32/18 LEU TTG ALA 19 GCG 1.4 ± 0.7 (5) 32/18 LEU TTG GLU 19 GAG 0.88 ± 0.37 (2) 33/19 PRO CC(T/C) LEU 19 CTG 0.13 (1) 33/19 PRO CC(T/C) GLN 19 CAG 0.22 ± 0.20 (2) 33/19 PRO CC(T/C) ALA 19 GCG 0.30 ± 0.14 (2) 33/19 PRO CC(T/C) THR 19 ACC <0.018 (1) 33/19 PRO CC(T/C) GLU 19 GAG 0.54 ± 0.43 (2) 34/20 LEU TTG VAL 19 GTG 1.2 ± 0.6 (3) 34/20 LEU TTG GLY 19 GGG 0.64 ± 0.74 (2) 34/20 LEU TTG SER 19 TCG 1.5 ± 0.7 (4) 34/20 LEU TTG LYS 19 AAG 0.97 ± 0.28 (2) 34/20 LEU TTG MET 19 ATG 1.1 ± 0.5 (3) 35/21 LEU CTG ALA 19 GCC 1.6 ± 0.5 (3) 35/21 LEU CTG GLY 19 GGC <0.006 ± 0.002 (3) 35/21 LEU CTG ASN 19 AAC 1.1 ± 1.7 (5) 35/21 LEU CTG PRO 19 CCC 1.8 ± 2.0 (5) 35/21 LEU CTG GLN 19 CAA 0.98 ± 1.1 (5) 35/21 LEU CTG VAL 19 GTG 0.76 ± 0.86 (5) 36/22 ASP GAC LEU 19 CTC 0.20 (1) 25/11 THR ACA ALA 19 GCC 0.86 ± 0.27 (3) 26/12 HIS CAC THR 19 ACG 0.010 (1) 26/12 HIS CAC PHE 19 TTC 0.26 (1) 26/12 HIS CAC GLY 19 GGG 0.19 (1) 26/12 HIS CAC ARG 19 CGG 0.21 (1) 26/12 HIS CAC ALA 19 GCC 0.56 ± 0.03 (2) 26/12 HIS CAC TRP 19 TGG — 27/13 LEU TTA GLY 19 GGG — 27/13 LEU TTA ARG 19 AGG — 27/13 LEU TTA THR 19 ATC 0.084 (1) 27/13 LEU TTA SER 19 TCC — 27/13 LEU TTA ALA 19 GCG 0.01 (1) 28/14 LYS AAG ARG 19 CGG 0.42 ± 0.07 (2) 28/14 LYS AAG LEU 19 TTG — 28/14 LYS AAG TRP 19 TGG — 29/14 LYS AAG GLN 19 CAG 0.27 (1) 28/14 LYS AAG GLY 19 GGC 0.36 ± 0.07 (2) 29/14 LYS AAG PRO 19 CCC 0.10 ± 0.04 (2) 28/14 LYS AAG VAL 19 GTG 0.19 ± 0.12 (2) 29/15 GLN CAG ASN 19 AAC 1.62 ± 1.7 (3) 29/15 GLN CAG LEU 19 CTG 0.284 29/15 GLN CAG PRO 19 CCG — 29/15 ARG CAG ARG 19 AGG 0.44 ± 0.16 (4) 29/15 GLN CAG VAL 19 GTG 0.62 ± 0.40 (4) 30/16 PRO CCA HIS 19 CAC 0.26 (1) 30/16 PRO CCA THR 19 ACG 0.36 (1) 30/16 PRO CCA GLY 19 GGG 1.2 ± 0.8 (3) 30/16 PRO CCA ASP 19 GAC — 30/16 PRO CCA GLN 19 CAG 0.61 ± 0.37 (3) 30/16 PRO CCA SER 19 TCG — 30/16 PRO CCA LEU 19 TTC — 30/16 PRO CCA LYS 19 AAG — 31/17 PRO CCT ASP 19 GAC 0.66 ± 0.71 (3) 36/22 ASP GAC VAL 19 GTG — 37/23 PHE TTC SER 19 AGC 0.62 ± 0.40 (4) 37/23 PHE TTC PRO 19 CCG 0.65 ± 0.39 (4) 37/23 PHE TTC TRP 19 TGG — 37/23 PHE TTC ILE 19 ATC 0.1 (1) 36/24 ASN AAC ALA 19 GCN 1.9 (1) 40/26 LEU CTC TRP 19 TGG — 40/26 LEU CTC ARG 19 CGC — 41/27 ASN AAT CYS 19 TGC 0.18 (1) 41/27 ASN AAT ARG 19 CGC 0.13 ± 0.13 (2) 41/27 ASN AAT LEU 19 CTG 0.09 ± 0.07 (2) 41/27 ASN AAT HIS 19 CAC 0.49 ± 0.26 (4) 41/27 ASN AAT MET 19 ATG 0.30 ± 0.38 (4) 41/27 ASN AAT PRO 19 CCG 0.12 (1) 42/28 GLY GGG ASP 19 GAC 5.7 ± 5.7 (6) 42/28 GLY GGG SER 19 AGC 4.3 ± 4.8 (7) 42/28 GLY GGG CYS 19 TGC 0.53 (1) 42/28 GLY GGG ALA 19 GCC 5.9 ± 4.1 (7) 43/29 GLU GAA ASN 19 AAC 0.050 (1) 43/29 GLU GAA TYR 19 TAC 0.010 (1) 43/29 GLU GAA LEU 19 CTC <0.009 (1) 43/29 GLU GAA PHE 19 TTC <0.009 (1) 43/29 GLU GAA ASP 19 GAC 0.044 (1) 43/29 GLU GAA ALA 19 GCC <0.009 (1) 43/29 GLU GAA CYS 19 TGC <0.009 (1) 43/29 GLU GAA SER 19 AGC <0.009 (1) 44/30 ASP GAC SER 19 TCA 0.007 (1) 44/30 ASP GAC LEU 19 CTG <0.007 (1) 44/30 ASP GAC ARG 19 AGG <0.007 (1) 44/30 ASP GAC LYS 19 AAG <0.007 (1) 44/30 ASP GAC THR 19 ACG — 44/30 ASP GAC MET 19 ATG <0.007 (1) 44/30 ASP GAC TRP 19 TGG <0.007 (1) 44/30 ASP GAC PRO 19 CCC <0.007 (1) 45/31 GLN CAA PRO 19 CCC — 45/31 GLN CAA PHE 19 TTC 0.007 (1) 45/31 GLN CAA VAL 19 GTC 6.7 ± 6.1 (5) 45/31 GLN CAA MET 19 ATG 3.4 ± 1.8 (5) 45/31 GLN CAA LEU 19 TTG 1.1 ± 1.3 (2) 45/31 GLN CAA THR 19 ACG 0.96 ± 1.5 (3) 45/31 GLN CAA LYS 19 AAG 1.6 ± 2.2 (5) 45/31 GLN CAA TRP 19 TGG 0.10 (1) 46/32 ASP GAC PHE 19 TTC 1.2 ± 0.5 (3) 46/32 ASP GAC SER 19 TCC 7.9 ± 6.4 (4) 46/32 ASP GAC THR 19 ACC 1.8 ± 0.2 (2) 46/32 ASP GAC CYS 19 TGC 0.80 (1) 46/32 ASP GAC GLY 19 GGC 0.25 (1) 47/33 ILE ATT GLY 19 GGC <0.015 (1) 47/33 ILE ATT VAL 19 GTG 0.38 (1) 47/33 ILE ATT HIS 19 CAC 0.10 (1) 47/33 ILE ATT SER 19 TCC 0.03 (1) 47/33 ILE ATT ARG 19 AGG 0.09 (1) 47/33 ILE ATT PRO 19 CCG <0.015 (1) 48/34 LEU CTG SER 19 AGC <0.009 (1) 48/34 LEU CTG CYS 19 TCG — 48/34 LEU CTG ARG 19 CGC <0.009 (1) 48/34 LEU CTG ILE 19 ATC 0.036 (1) 48/34 LEU CTG HIS 19 CAC <0.009 (1) 48/34 LEU CTG PHE 19 TTC <0.009 (1) 48/34 LEU CTG ASN 19 AAC <0.009 (1) 49/35 MET ATG ARG 19 CGC 0.007 (1) 49/35 MET ATG ALA 19 GCC 0.091 (1) 49/35 MET ATG GLY 19 GGC 0.036 (1) 49/35 MET ATG PRO 19 CCC <0.009 (1) 49/35 MET ATG ASN 19 AAC 0.23 (1) 49/35 MET ATG HIS 19 CAC <0.009 (1) 49/35 MET ATG ASP 19 GAC 0.28 ± 0.48 (3) 50/36 GLU GAA LEU 19 CTC 0.01 (1) 50/36 GLU GAA THR 19 ACC 0.20 (1) 50/36 GLU GAA ASP 19 GAC — 50/36 GLU GAA TYR 19 TAC 0.09 (1) 50/36 GLU GAA GLN 19 CTG 0.02 (1) 51/37 ASN AAT ARG 19 CGC 2.0 ± 0.8 (3) 51/37 ASN AAT MET 19 ATG 0.75 ± 0.50 (2) 51/37 ASN AAT PRO 19 CCG 2.77 ± 1.6 (3) 51/37 ASN AAT SER 19 TCC 0.87 ± 0.44 (3) 51/37 ASN AAT THR 19 ACG 2.3 ± 1.6 (3) 51/37 ASN AAT HIS 19 CAC 1.3 ± 0.9 (5) 52/38 ASN AAC HIS 19 CAC 0.004 (1) 52/39 ASN AAC ARG 19 CGC 0.004 (1) 52/36 ASN AAC LEU 19 TGG 0.003 (1) 52/38 ASN AAC GLY 19 GGC 0.22 (1) 52/38 ASN AAC SER 19 AGC 0.07 (1) 52/38 ASN AAC THR 19 ACG 0.44 ± 0.30 (3) 53/39 LEU CTT THR 19 ACC <0.005 (1) 53/39 LEU CTT ALA 19 GCG — 53/39 LEU CTT GLY 19 GGC <0.005 (1) 53/39 LEU CTT GLU 19 GAG <0.005 (1) 53/39 LEU CTT PRO 19 CCG <0.005 (1) 53/39 LEU CTT LYS 19 AAG <0.005 (1) 53/39 LEU CTT SER 19 AGC 0.008 (1) 53/39 LEU CTT MET 19 ATG 0.31 (1) 54/40 ARG CGA ASP 19 GAC <0.005 (1) 54/40 ARG CGA ILE 19 ATC 0.05 (1) 54/40 ARG CGA SER 19 TCC 0.10 (1) 54/40 ARG CGA VAL 19 GTC <0.005 (1) 54/40 ARG CGA THR 19 ACC 0.015 (1) 54/40 ARG CGA GLN 19 CAG 0.04 (1) 54/40 ARG CGA LEU 19 TTG 0.03 (1) 55/41 ARG AGC THR 19 ACC 0.65 ± 1.1 (4) 55/41 ARG AGG VAL 19 GTC 0.96 ± 0.36 (3) 55/41 ARG AGG SER 19 TCG 0.065 (1) 55/41 ARG AGG LEU 19 CTG 1.1 ± 1.2 (4) 55/41 ARG AGC GLY 19 GGC 1.0 ± 0.6 (4) 56/42 PRO CCA GLY 19 GGC 1.1 ± 0.8 (3) 56/42 PRO CCA CYS 19 TGC 0.21 (1) 56/42 PRO CCA SER 19 AGC 1.4 ± 0.4 (2) 56/42 PRO CCA GLN 19 CAG 1.8 (1) 56/42 PRO CCA LYS 19 AAG 0.60 (1) 57/43 ASN AAC GLY⁶ 19 GGC — 58/44 LEU CTG SER 19 ACC <0.041 (1) 58/44 LEU CTG ASP 19 GAC <0.041 (1) 58/44 LEU CTG ARG 19 CGG <0.041 (1) 58/44 LEU CTG GLN 19 CAG <0.041 (1) 58/44 LEU CTG VAL 19 GTC <0.041 (1) 58/44 LEU CTG CYS 19 TGC — 59/45 GLU GAG TYR 19 TAC 0.41 ± 0.37 (5) 59/45 GLU GAG HIS 19 CAC 0.38 ± 0.31 (2) 59/45 GLU GAG LEU 19 CTC 0.46 ± 0.36 (6) 59/45 GLU GAG PRO 19 CCC — 59/45 GLU GAG ARG 19 CGC 0.15 (1) 60/46 ALA GCA SER 19 AGC 0.91 ± 0.55 (4) 60/46 ALA GCA PRO 19 CCC — 60/46 ALA GCA TYR 19 TAC <0.008 (1) 60/46 ALA GCA ASN 19 AAC 0.38 (1) 60/46 ALA GCA THR 19 ACG 0.21 (1) 61/47 PHE TTC ASN 19 AAC — 61/47 PHE TTC GLU 19 GAG <0.010 (1) 61/47 PHE TTC PRO 19 CCC — 61/47 PHE TTC LYS 19 AAG <0.010 (1) 61/47 PHE TTC ARG 19 CGC 0.006 (1) 61/47 PHE TTC SER 19 TCG 0.17 (1) 62/48 ASN AAC HIS 19 CAC — 62/48 ASN AAC VAL 19 GTG 0.37 ± 0.25 (4) 62/48 ASN AAC ARG 19 AGG — 62/48 ASN AAC PRO⁷ 19 CCG 1.6 ± 0.4 (3) 62/48 ASN AAC PRO 19 CCG 2.0 ± 0.3 (3) 62/48 ASN AAC THR⁸ 19 ACG 2.3 ± 1.1 (3) 62/45 ASN AAC ASP 19 GAC — 62/48 ASN AAC ILE 19 ATC 0.56 ± 0.24 (4) 63/49 ARG A(G/A)G TYR 19 TAC 0.47 (1) 63/49 ARG A(G/A)G TRP 19 TGG 0.09 (1) 63/49 ARG A(G/A)G LYS 19 AGG 0.52 (1) 63/49 ARG A(G/A)G SER⁹ 19 TCC 0.13 (1) 63/49 ARG A(G/A)G HIS 19 CAC 0.42 ± 0.25 (7) 63/49 ARG A(G/A)G PRO 19 CCG <0.014 ± 0.013(2) 63/49 ARG A(G/A)G VAL 19 GTG 0.39 ± 0.34 (3) 64/50 ALA GCT ASN 19 AAC 1.5 ± 2.9 (4) 64/50 ALA GCT PRO 19 CCG <0.023 (1) 64/50 ALA GCT SER 19 AGC <0.023 (1) 64/50 ALA GCT LYS 19 AAG <0.047 (1) 65/51 VAL GTC THR 19 ACC 0.71 ± 0.64 (3) 65/51 VAL GTC PRO 19 CCG <0.014 (1) 65/51 VAL GTC HIS 19 CAC <0.014 (1) 65/51 VAL GTC LEU 19 CTC 0.42 (1) 65/51 VAL GTC PHE 19 TTC 0.061 (1) 65/51 VAL GTC SER 19 TCC 0.34 (1) 66/52 LYS AAG ILE¹⁰ 19 ATC 0.42 (1) 66/52 LYS AAG ARG 19 AGG 0.79 ± 0.18 (2) 66/52 LYS AAG VAL 19 GTC 0.38 ± 0.17 (2) 66/52 LYS AAG ASN 19 AAC 0.32 (1) 66/52 LYS AAG GLU 19 GAG 0.14 (1) 66/52 LYS AAG SER 19 TCG 0.31 (1) 66/52 LYS AAG VAL¹¹ 19 GTG 0.055 (1) 67/53 SER AGT ALA 19 GCG <0.014 (1) 67/53 SER AGT PHE 19 TTC 1.2 ± 0.2 (2) 67/53 SER AGT VAL 19 GTG 0.24 (1) 67/53 SER AGT GLY 19 GGG 0.50 ± 0.29 (4) 67/53 SER AGT ASN 19 AAC 0.52 ± 0.28 (7) 67/53 SER AGT ILE 19 ATC 0.29 (1) 67/53 SER AGT PRO 19 CCG 0.055 (1) 67/53 SER AGT HIS 19 CAC 0.99 ± 0.62 (6) 68/54 LEU TTA VAL 19 GTC 0.14 (1) 68/54 LEU TTA TRP 19 TGG 0.07 (1) 68/54 LEU TTA SER 19 AGC <0.003 (1) 68/54 LEU TTA ILE 19 ATC 0.84 ± 0.47 (3) 68/54 LEU TTA PHE 19 TTC 1.7 ± 0.3 (3) 68/54 LEU TTA THR 19 ACG 0.011 (1) 68/54 LEU TTA HIS 19 CAC 0.82 ± 0.45 (2) 69/55 GLN CAG ALA 19 GCG 1.2 ± 0.8 (3) 69/55 GLN CAG PRO 19 CCA 0.74 0.45 (4) 69/55 GLN CAG THR 19 ACG 0.97 ± 0.46 (4) 69/55 GLN CAG TRP 19 TGG — 69/55 GLN CAG GLU 19 GAG 1.4 ± 0.7 (3) 69/55 GLN CAG ARG 19 CGG 1.4 ± 1.1 (3) 69/55 GLN CAG GLY 19 GGG 0.68 ± 0.02 (2) 69/55 GLN CAG LEU 19 CTC — 70/56 ASN AA(C/T) LEU 19 TTG 0.032 (1) 70/56 ASN AA(C/T) VAL 19 GTG — 70/56 ASN AA(C/T) TRP 19 TGG — 70/56 ASN AA(C/T) PRO¹² 19 CCG 0.43 ± 0.29 (2) 70/56 ASN AA(C/T) ALA¹³ 19 GCC 0.03 (1) 71/57 ALA GCA MET 19 ATG 0.23 (1) 71/57 ALA GCA LEU 19 CTG <0.005 (1) 71/57 ALA GCA PRO 19 CCC 0.58 (1) 71/57 ALA GCA ARG 19 AGG 0.66 (1) 71/57 ALA GCA GLU 19 GAG 0.46 ± 0.27 71/57 ALA GCA THR 19 ACC 0.34 ± 0.41 (3) 71/57 ALA GCA GLN 19 GGC 0.42 ± 0.32 (3) 71/57 ALA GCA TRP 19 TGG — 71/57 ALA GCA ASN 19 AAC 0.09 (1) 72/58 SER TCA GLU 19 GAG 0.62 ± 0.27 (3) 72/56 SER TCA MET 19 ATG 0.45 ± 0.55 (3) 72/58 SER TCA ALA 19 GCC 0.48 ± 0.33 (3) 72/58 SER TCA HIS 19 CAC 0.10 (1) 72/58 SER TCA ASN 19 AAC 0.38 ± 0.44 (3) 72/58 SER TCA ARG 19 CGG 0.81 ± 0.43 (4) 72/58 SER TCA ASP 19 GAC 0.58 ± 0.39 (3) 73/59 ALA GCA GLU 19 GAG 0.49 ± 0.32 (3) 73/59 ALA GCA ASP 19 GAC 0.27 (1) 73/59 ALA GCA LEU 19 CTG 0.55 ± 0.45 (4) 73/59 ALA GCA SER 19 AGC 0.37 ± 0.36 (2) 73/59 ALA GCA GLY 19 CGG 0.38 ± 0.32 (3) 73/59 ALA GCA THR 19 ACC 0.31 (1) 73/59 ALA GCA ARG 19 AGG 0.40 ± 0.18 (3) 74/60 ILE AT(T/C) MET 19 ATG <0.16 (1) 74/60 ILE AT(T/C) THR 19 ACG — 74/60 ILE AT(T/C) PRO 19 CCG — 74/60 ILE AT(T/C) ARG 19 AGG — 74/60 ILE AT(T/C) GLY 19 GCG 0.006 (1) 74/60 ILE AT(T/C) ALA 19 GCG — 75/61 GLU GAG LYS 19 AAG 0.07 ± 0.07 (2) 75/61 GLU GAG GLY 19 GGG 0.27 ± 0.20 (2) 75/61 GLU GAG ASP 19 GAC 0.18 (1) 75/61 GLU GAG PRO 19 CCG — 75/61 GLU GAG TRP 19 TGG — 75/61 GLU GAG ARG 19 CGG — 75/61 GLU GAG SER 19 TCG 0.27 ± 0.22 (3) 75/61 GLU GAG GLN 19 CAG 0.40 ± 0.38 (3) 75/61 GLU GAG LEU 19 TTG — 76/62 SER AGC VAL 19 GTG 1.0 ± 0.2 (2) 76/62 SER AGC ALA 19 GCG 0.94 ± 0.46 (2) 76/62 SER AGC ASN 19 AAC 1.2 (1) 76/62 SER AGC TRP 19 TGG — 76/62 SER AGC GLU 19 GAG 0.90 ± 0.19 (2) 76/62 SER AGC PRO 19 CCG 2.1 ± 0.8 (4) 76/62 SER AGC GLY 19 GGC 1.3 ± 1.0 (4) 76/62 SER AGC ASP 19 GAC 0.29 (1) 77/63 ILE ATT SER 19 AGC 0.48 ± 0.38 (4) 77/63 ILE ATT ARG 19 CGC 0.09 ± 0.04 (2) 77/63 ILE ATT THR 19 ACG <0.008 (1) 77/63 ILE ATT LEU 19 TTG 2.0 ± 0.1 (3) 78/64 LEU CTT ALA 19 GCG — 78/64 LEU CTT SER 19 TCC — 78/64 LEU CTT GLU 19 GAG <0.006 (1) 78/64 LEU CTT PHE 19 TTC — 78/64 LEU CTT GLY 19 GGG — 78/64 LEU CTT ARG 19 AGG — 79/65 LYS AA(A/G) THR 19 ACA 0.77 ± 0.91 (6) 79/65 LYS AA(A/G) GLY 19 GGG 1.1 ± 0.9 (6) 79/65 LYS AA(A/G) ASN 19 AAC 1.0 ± 0.6 (6) 79/65 LYS AA(A/G) MET 19 ATG 1.6 ± 0.7 (6) 79/65 LYS AA(A/G) ARG 19 CGC 1.04 ± 0.7 (7) 79/65 LYS AA(A/G) ILE 19 ATC 1.0 ± 0.6 (6) 79/65 LYS AA(A/G) GLY 19 GGG 1.2 ± 0.4 (6) 79/65 LYS AA(A/G) ASP 19 GAC 0.72 ± 0.38 (7) 80/66 ASN AAT TRP 19 TGG — 80/66 ASN AAT VAL 19 GTC 0.32 (1) 80/66 ASN AAT GLY 19 GGC 1.5 ± 1.4 (4) 80/66 ASN AAT THR 19 ACG 0.13 (1) 80/66 ASN AAT LEU 19 CTG 0.33 ± 0.14 (2) 80/66 ASN AAT GLU 19 GAG 1.1 ± 0.8 (4) 80/66 ASN AAT ARG 19 AGG 1.0 ± 0.8 (4) 81/67 LEU CTC GLN 19 CAA — 81/67 LEU CTC GLY 19 GGC <0.023 (1) 81/67 LEU CTC ALA 19 GCG <0.047 (1) 81/67 LEU CTC TRP 19 TGG <0.005 (1) 81/67 LEU CTC ARG 19 CGG — 81/67 LEU CTC VAL 19 GTG 0.16 ± 0.18 (2) 81/67 LEU CTC LYS 19 AAG — 82/68 LEU C(TG/CC) GLN 19 CAG 1.8 ± 0.3 (3) 82/68 LEU C(TG/CC) LYS 19 AAG 0.05 (1) 82/68 LEU C(TG/CC) TRP 19 TGG 2.7 ± 1.3 (4) 82/68 LEU C(TG/CC) ARG 19 AGC 1.1 ± 0.2 (3) 82/68 LEU C(TG/CC) ASP 19 GAC 2.7 ± 1.3 (4) 82/68 LEU C(TG/CC) VAL 19 GTG 1.5 ± 1.1 (5) 83/69 PRO CCA ALA 19 GCA 0.41 (1) 83/69 PRO CCA THR 19 ACC 0.66 ± 0.12 (3) 83/69 PRO CCA ARG 19 CGG — 83/69 PRO CCA TRP 19 TGG 0.29 (1) 83/69 PRO CCA MET 19 ATG 0.43 ± 0.28 (3) 84/70 CYS TG(T/C) GLU 19 GAG <0.014 (1) 84/70 CYS TG(T/C) GLY 19 GGG <0.006 (1) 84/70 CYS TG(T/C) ARG 19 AGG — 84/70 CYS TG(T/C) MET 19 ATG — 84/70 CYS TG(T/C) VAL 19 GTG — 85/71 LEU CTG ASN 19 AAC — 85/71 LEU CTG VAL 19 GTG 0.52 ± 0.21 (5) 85/71 LEU CTG GLN 19 CAG — 86/72 PRO CCC CYS 19 TGC — 86/72 PRO CCC ARG 19 AGG — 86/72 PRO CCC ALA 19 GCG — 86/72 PRO CCC LYS 19 AAG — 87/73 LEU (C/A)TG SER 19 AGC 1.5 ± 0.4 (3) 87/73 LEU (C/A)TG TRP 19 TGG — 87/73 LEU (C/A)TG GLY 19 GGG — 88/74 ALA GCC LYS 19 AAG — 88/74 ALA GCC ARG 19 AGG 0.11 ± 0.10 (2) 88/74 ALA GCC VAL 19 GTG 0.09 ± 0.02 (2) 88/74 ALA GCC TRP 19 TGG 1.8 ± 0.2 (2) 89/75 THR AC(G/A) ASP 19 GAC 0.24 ± 0.10 (2) 89/75 THR AC(G/A) CYS 19 TGC — 89/75 THR AC(G/A) LEU 19 CTC 0.01 (1) 89/75 THR AC(G/A) VAL 19 GTG 0.08 (1) 69/75 THR AC(G/A) GLU 19 GAG 0.11 (1) 89/75 THR AC(G/A) HIS 19 CAC 0.16 ± 0.06 (2) 89/75 THR AC(G/A) ASN 19 AAC 0.21 ± 0.04 (2) 89/75 THR AC(G/A) SER 19 TCG 0.25 ± 0.07 (2) 90/76 ALA GCC PRO 19 CCC 0.03 (1) 90/76 ALA GCC SER 19 TCG — 90/76 ALA GCC THR 19 ACC 0.48 (1) 90/76 ALA GCC GLY 19 GGC <0.006 (1) 90/76 ALA GCC ASP 19 GAC 0.44 ± 0.29 (4) 90/76 ALA GCC ILE 19 ATC — 90/76 ALA GCC MET 19 ATG 0.25 ± 0.13 (2) 91/77 ALA GCA PRO 19 CCC 1.9 ± 1.2 (3) 91/77 ALA GCA SER 19 TCC 0.12 ± 0.07 (2) 91/77 ALA GCA THR 19 ACC 0.48 ± 0.16 (2) 91/77 ALA GCA PHE 19 TTC 0.44 ± 0.50 (3) 91/77 ALA GCA LEU 19 CTC 0.43 ± 0.27 (S) 91/77 ALA GCA ASP 19 GAC 0.55 ± 0.09 (2) 91/77 ALA GCA HIS 19 CAC — 92/76 PRO CCC PHE 19 TTC — 92/78 PRO CCC ARG 19 CGG — 92/78 PRO CCC SER 19 AGC 0.26 (1) 92/78 PRO CCC LYS 19 AAG — 92/78 PRO CCC HIS 19 CAC — 92/78 PRO CCC LEU 19 CTG — 93/79 THR ACG ASP 19 GAC 1.3 ± 0.7 (4) 93/79 THR ACG SER 19 TCG 0.70 ± 0.56 (4) 93/79 THR ACG ASN 19 AAC — 93/79 THR ACG PRO 19 CCC 0.53 ± 0.36 (4) 93/79 ThR ACG ALA 19 GCG 1.13 ± 0.2 (3) 93/79 THR ACG LEU 19 CTG 0.69 ± 0.42 93/79 THR ACG ARG 19 CGC 0.93 ± 0.96 (4) 94/80 ARG CGA ILE 19 ATC <0.020 (1) 94/80 ARG CGA SER 19 TCC <0.100 (1) 94/80 ARG CGA GLU 19 GAG <0.020 (1) 94/90 ARG CGA LEU 19 CTG <0.020 (1) 94/50 ARG CGA VAL 19 GTG <0.024 (1) 94/80 ARG CGA PRO 19 CCC <0.024 (1) 95/81 HIS CAT GLN 19 CAG <0.010 (1) 95/81 HIS CAT PRO 19 CCG 1.6 ± 0.8 (3) 95/81 HIS CAT ARG 19 CGC 4.7 ± 5.9 (2) 95/81 HIS CAT VAL 19 GTC 1.2 ± 1.7 (2) 95/81 HIS CAT LEU 19 CTC 0.7 (1) 95/81 HIS CAT GLY 19 GGC 1.7 ± 2.4 (5) 95/81 HIS CAT THR 19 ACC 2.9 ± 4.5 (4) 95/81 HIS CAT TYR 19 TAC 0.07 (1) 96/82 PRO CCA LYS 19 AAG <0.010 ± 0.001 (2) 96/82 PRO CCA TYR 19 TAC 0.69 (1) 96/82 PRO CCA GLY 19 GGG <0.040 (1) 96/82 PRO CCA ILE 19 ATC <0.040 (1) 96/82 PRO CCA THR 19 ACC <0.040 (1) 97/83 ILE ATC VAL 19 GTC 0.91 ± 1.2 (8) 97/83 ILE ATC LYS 19 AAG <0.024 97/83 ILE ATC ALA 19 GCG 0.15 (1) 97/83 ILE ATC ASN 19 AAT <0.02 (1) 98/84 HIS CAT ILE 19 ATC 5.0 ± 4.9 (12) 98/84 HIS CAT ASN 19 AAC 1.4 ± 0.4 (2) 98/84 HIS CAT LEU 19 CTC 2.4 ± 1.0 (2) 98/84 HIS CAT ASP 19 GAC 0.38 ± 0.49 (5) 98/84 HIS CAT ALA 19 GCC 2.0 ± 1.0 (3) 98/84 HIS CAT THR 19 ACG 1.6 ± 0.3 (2) 98/84 HIS CAT LEU 19 TTG 1.5 (1) 98/84 HIS CAT PRO 19 CCG 0.55 (1) 99/85 ILE ATC LEU 19 CTG 1.4 ± 1.4 (7) 99/85 ILE ATC ARG 19 CGC <0.025 (1) 99/85 ILE ATC ASP 19 GAC <0.025 (1) 99/65 ILE ATC VAL 19 GTC 0.51 ± 0.59 (3) 99/55 ILE ATC PRO 19 CCG <0.025 (1) 99/85 ILE ATC GLN 19 CAG <0.018 ± 0.010 (2) 99/85 ILE ATC GLY 19 GGG <0.018 ± 0.10 (2) 99/85 ILE ATC SER 19 TCG <0.025 (1) 99/85 ILE ATC PHE 19 TTC 0.45 (1) 99/85 ILE ATC HIS 19 CAC <0.025 (1) 100/86 LYS AAG TYR 19 TAC 0.03 (1) 100/96 LYS AAG LEU 19 TTG 0.33 ± 0.31 (3) 100/96 LYS AAG HIS 19 CAC 0.36 ± 0.22 (9) 100/86 LYS AAG ARG 19 AGC 4.7 ± 5.9 (4) 100/86 LYS AAG ILE 19 ATC 0.95 (1) 100/86 LYS AAG SER 19 AGC 0.95 (1) 100/86 LYS AAG GLN 19 CAG 0.78 ± 0.80 (7) 100/86 LYS AAG PRO 19 CCG 0.70 (1) 101/87 ASP GAC PRO 19 CCC 2.3 ± 3.1 (4) 101/87 ASP GAC MET 19 ATG 1.8 ± 2.5 (6) 101/87 ASP GAC LYS 19 AAG 1.2 ± 1.7 (3) 101/87 ASP GAC HIS 19 CAC 2.5 (1) 101/87 ASP GAC THR 19 ACG 0.90 ± 0.77 (3) 101/87 ASP GAC TYR 19 TAC 0.59 (1) 101/87 ASP GAC VAL 19 GTC 0.42 (1) 101/87 ASP GAC TYR 19 TAC 1.0 ± 0.02 (2) 101/87 ASP GAC GLN 19 CAG 0.07 (1) 102/88 GLY GGT LEU 19 CTC <0.015 ± 0.007 (2) 102/88 GLY GGT GLU 19 GAG 0.40 ± 0.07 (3) 102/88 GLY GGT LYS 19 AGG 0.16 ± 0.14 (2) 102/88 GLY GGT SER 19 TCC 0.29 (1) 102/88 GLY GGT TYR 19 TAC 0.04 (1) 102/88 GLY GGT PRO 19 CCC <0.011 (1) 103/89 ASP GAC SER 19 TCC 0.02 (1) 104/90 TRP TGG VAL 19 GTG 0.11 ± 0.06 (5) 104/90 TRP TGG CYS 19 AGC 0.07 ± 0.03 (5) 104/90 TRP TGG TYR 19 TAC 0.34 ± 0.42 (5) 104/90 TRP TGG THR 19 ACC 0.04 ± 0.02 (2) 104/90 TRP TGG MET 19 ATG 0.14 (1) 104/90 TRP TGG PRO 19 CCC 0.02 ± 0.02 (2) 104/90 TRP TGG LEU 19 TTG 0.65 ± 1.0 (3) 104/90 TRP TGG GLN 19 CAG 0.008 (1) 104/90 TRP TGG LYS 19 AAG — 104/90 TRP TGG GLY 19 GAG — 104/90 TRP TGG ALA 19 GCC — 104/90 TRP TGG PHE 19 TTC — 104/90 TRP TGG GLY 19 GGC — 105/91 ASN AAT PRO 19 CCG 4.8 ± 8.5 (5) 105/91 ASN AAT ALA 19 GCC 0.65 ± 0.30 (3) 105/91 ASN AAT PHE 19 TTC 0.13 (1) 105/91 ASN AAT SER 19 TCC 1.9 ± 2.7 (5) 105/91 ASN AAT TRP 19 TGG 0.95 (1) 105/91 ASN AAT GLN 19 CAA 0.57 ± 0.52 (3) 105/91 ASN AAT TYR 19 TAC 0.66 ± 0.53 (4) 105/91 ASN AAT LEU 19 CTC 0.87 ± 0.79 (2) 105/91 ASN AAT LYS 19 AAG 0.70 (1) 105/91 ASN AAT ILE 19 ATC 1.0 (1) 105/91 ASN AAT ASP 19 GAC 1.0 ± 0.9 (4) 105/91 ASN AAT HIS 19 CAC 0.71 ± 0.48 (2) 106/92 GLU GAA SER 19 TCC 0.17 ± 0.21 (2) 106/92 GLU GAA ALA 19 GCG 0.235 ± 0.26 (2) 106/92 GLU GAA LYS 19 AAG — 106/92 GLU GAA THR 19 ACC — 106/92 GLU GAA ILE 19 ATC — 106/92 GLU GAA GLY 19 GGC 0.70 ± 0.76 (4) 106/92 GLU GAA PRO 19 CCC — 108/94 ARG CGG LYS 19 AAG 0.11 ± 0.03 (2) 108/94 ARG CGG ASP 19 GAC — 108/94 ARG CGG LEU 19 TTG 0.01 (1) 108/94 ARG CGG THR 19 ACG 0.08 (1) 108/94 ARG CGG ILE 19 ATC <0.01 (1) 108/94 ARG CGG PRO 19 CCC — 109/95 ARG AGG THR 19 ACC 1.1 ± 0.2 (3) 109/95 ARG AGG PRO 19 CCC — 109/95 ARG AGG GLU 19 GAG 1.1 ± 0.1 (3) 109/95 ARG AGG TYR 19 TAC <0.006 (1) 109/95 ARG AGG LEU 19 CTC 1.2 ± 0.9 (4) 109/95 ARG AGG SER 19 TCG 1.7 ± 0.8 (4) 109/95 ARG AGG GLY 19 GGG 0.17 (1) 110/96 LYS AAA ALA 19 GCC <0.08 (1) 110/96 LYS AAA ASN 19 AAC — 110/96 LYS AAA THR 19 ACG — 110/96 LYS AAA LEU 19 CTC — 110/96 LYS AAA ARG 19 CGG — 110/96 LYS AAA GLN 19 CAG — 110/96 LYS AAA TRP 19 TGG — 111/97 LEU CTG ILE 19 ATC — 111/97 LEU CTG ARG 19 CGG — 111/97 LEU CTG ASP 19 GAC — 111/97 LEU CTG MET 19 ATG — 112/98 THR ACG VAL 19 GTG 0.55 ± 0.44 (3) 112/98 THR ACG GLN 19 CAG 1.7 ± 1.0 (3) 112/98 THR ACG TYR 19 TAC <0.018 (1) 112/98 THR ACG GLU 19 GAG 0.12 (1) 112/98 THR ACG HIS 19 CAC 0.25 ± 0.40 (3) 112/99 THR ACG SER 19 TCC 0.17 ± 0.15 (2) 112/98 THR ACG PHE 19 TTC — 113/99 PHE TTC SER 19 AGC — 113/99 PHE TTC CYS 19 TGC — 113/99 PHE TTC HIS 19 CAC <0.009 (1) 113/99 PHE TTC GLY 19 GGC — 113/99 PHE TTC TRP 19 TGG — 113/99 PHE TTC TYR 19 TAC 0.07 (1) 113/99 PHE TTC ASN 19 AAC — 114/100 TYR TAT CYS 19 TGC — 114/100 TYR TAT HIS 19 CAC — 114/100 TYR TAT SER 19 AGC — 114/100 TYR TAT TRP 19 TGG 0.88 (1) 114/100 TYR TAT ARG 19 AGG — 114/100 TYR TAT LEU 19 CTC <0.018 (1) 115/101 LEU CTG ASN 19 AAC <0.004 (1) 115/101 LEU CTG VAL 19 GTG — 115/101 LEU CTG PRO 19 CCC <0.004 (1) 115/101 LEU CTG ARG 19 AGG <0.004 (1) 115/101 LEU CTG ALA 19 GCG 0.50 (1) 115/101 LEU CTG HIS 19 CAC — 115/101 LEU CTG THR 19 ACC — 115/101 LEU CTG TRP 19 TGG — 115/101 LEU CTG MET 19 ATG <0.008 (1) 116/102 LYS AAA LEU¹⁴ 19 TTG — 116/102 LYS AAA PRO¹⁴ 19 CCG <0.004 (1) 116/102 LYS AAA THR¹⁴ 19 ACC 0.50 (1) 116/102 LYS AAA MET¹⁴ 19 ATG 0.13 (1) 116/102 LYS AAA ASP¹⁴ 19 GAC <0.018 (1) 116/102 LYS AAA VAL 19 GTG 2.3 ± 1.2 (5) 116/102 LYS AAA GLU 19 GAG 0.06 (1) 116/102 LYS AAA ARG 19 CGC 0.06 (1) 116/102 LYS AAA TRP 19 TGG 2.3 ± 1.0 (4) 116/102 LYS AAA SER 19 TCG 0.69 ± 0.51 (5) 116/102 LYS AAA LEU 19 CTC 0.14 ± 0.02 (2) 116/102 LYS AAA ILE 19 ATC 1.3 ± 0.3 (3) 116/102 LYS AAA THR 19 ACG 0.84 ± 0.30 (4) 117/103 THR ACC SER 19 AGC 1.1 ± 0.2 (3) 117/103 THR ACC ASN 19 AAC 0.31 ± 0.39 (3) 117/103 THR ACC ILE 19 ATC — 117/103 THR ACC TRP 19 TGG 0.02 (1) 117/103 THR ACC LYS 19 AAG <0.005 (1) 117/103 THR ACC PRO 19 CCG — 118/104 LEU CTT SER 19 TCA — 118/104 LEU CTT PRO 19 CCC — 118/104 LEU CTT ALA 19 GCC — 118/104 LEU CTT GLU 19 GAG — 118/104 LEU CTT CYS 19 TGC — 118/104 LEU CTT ASP 19 GAC — 118/104 LEU CTT TYR 19 TAC — 119/105 GLU GAG SER 19 TCC 0.26 ± 0.19 (2) 119/105 GLU GAG LYS 19 AAG 0.04 (1) 119/105 GLU GAG PRO 19 CCG 0.31 ± 0.27 (3) 119/105 GLU GAG LEU 19 CTG 0.35 ± 0.35 (3) 119/105 GLU GAG THR 19 ACC 0.25 ± 0.27 (3) 119/105 GLU GAG TYR 19 TAC 0.30 ± 0.32 (3) 119/105 GLU GAG ARG 19 CGC 0.06 (1) 120/106 ASN AAT ALA 19 GCC <0.009 (1) 120/106 ASN AAT PRO 19 CCC 1.7 ± 0.7 (3) 120/106 ASN AAT LEU 19 TTG 1.2 ± 0.3 (3) 120/106 ASN AAT HIS 19 CAC 1.0 ± 0.3 (2) 120/106 ASN AAT VAL 19 GTG 1.7 ± 0.3 (3) 120/106 ASN AAT GLN 19 CAG 0.85 ± 0.16 (2) 121/107 ALA GCG SER 19 AGC 1.2 ± 0.2 (3) 121/107 ALA GCG ILE 19 ATC 2.8 ± 2.5 (2) 121/107 ALA GCG ASN 19 AAC 0.91 ± 0.77 (5) 121/107 ALA GCG PRO 19 CCG 1.3 (1) 121/107 ALA GCG LYS 19 AAG 0.26 ± 0.24 (2) 121/107 ALA GCG ASP 19 GAC 1.8* ± 0.9 (3) 121/107 ALA GCG GLY 19 GGC 0.69 (1) 122/108 GLN GCG SER 19 AGC 0.9.6 ± 0.41 (3) 122/108 GLN CA(G/A) MET 19 ATG 1.7 ± 0.5 (3) 122/108 GLN CA(G/A) TRP 19 TGG 1.4 (1) 122/108 GLN CA(G/A) ARG 19 AGG 0.78 (1) 122/108 GLN CA(G/A) PHE 19 TTC 2.3 ± 1.1 (3) 122/108 GLN CA(G/A) PRO 19 CCG 1.0 (1) 122/108 GLN CA(G/A) HIS 19 CAC 1.4 (1) 122/108 GLN CA(G/A) ILE 19 ATC 2.7 ± 0.8 (3) 122/108 GLN CA(G/A) TYR 19 TAC 1.7 ± 0.3 (2) 122/108 GLN CA(G/A) CYS 19 TGC 0.58 (1) 123/109 ALA GCT MET 19 ATG 2.0 ± 0.2 (3) 123/109 ALA GCT GLU 19 GAG 2.1 ± 1.0 (3) 123/109 ALA GCT HIS 19 CAC 0.98 ± 0.72 (3) 123/109 ALA GCT SER 19 AGC 1.4 ± 0.8 (3) 123/109 ALA GCT PRO 19 CCC 0.64 ± 0.16 (2) 123/109 ALA GCT TYR 19 TAC 0.51 ± 0.25 (2) 123/109 ALA GCT LEU 19 CTG 1.2 ± 0.1 (2) ¹The first position number representes the amino acid position in (1-133)hIL-3 and the second number represents the position in (15-125)hIL-3 in which the Asn at position 15 of native hIL-3 is position 1 in (15-125)hIL-3 (See the numbering for Formula XI) ²Double mutant; has PRO at position 35. ³Double mutant; has THR at position 49. ⁴Double mutant; has Gly at position 32. ⁵Double mutant; has Leu at position 31. ⁶Double mutant; has Gly at position 46. ⁷Double mutant; Arg at position 42. ⁸Double mutant; Phe at position 53. ⁹Double mutant; has Val at position 49. ¹⁰Double mutant; has Pro at position 73. ¹¹Double mutant; has Thr at position 64. ¹²Double mutant; has Pro at position 73. ¹³Double mutant; has Met at position 74. ¹⁴Double mutant; has Ser at position 105.

The mutants in Table 6 were made as described in the Examples, particularly Examples 19, 20, 21 and 38 to 53.

It will be apparent to those skilled in the art that other codons besides those shown in Table 6 can also code for the substituted amino acids in the hIL-3 muteins. The present invention includes the DNAs encoding the mutant hIL-3 polypeptides of the invention including the various codons which can code for the parental and substituted amino acids of the hIL-3 muteins of the invention due to the degeneracy of the genetic code.

hIL-3 (15-125) variant genes encoding the variants listed in Table 6 can also be expressed from intracellular expression vectors to produce large quantities of the variant protein which can be purified and assayed for biological activity. The hIL-3 variant genes, from Table 6, can be excised from the secretion expression vector, as a 345 base pair NcoI/HindIII fragment and ligated into an appropriate intracellular expression vector, such as pMON2341 digested with NcoI and HindIII. Examples of variants transferred to pMON2341 in this manner are shown in Table 7. Two examples of such a transfer are described. in the construction of pMON13215 (EXAMPLE 64) and pMON13252 (EXAMPLE 65).

EXAMPLE 64

Construction of pMON13215

Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication, F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Trp⁽¹¹⁶⁾ variant, from Table 6 wag digested with NcoT and HindIII resulting in a 345 base pair NcoI/HindIII fragment. The 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform E.coli K-12 strain JM101. Plasmid DNA was isolated and screened by restriction anaylsis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated PMON132I5. The plasmid, pMON13215, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

PEPTIDE A9; (15-125)HIL-3 TRP⁽¹¹⁶⁾ PMON13215

        Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu [SEQ ID NO:217]         15                  20                  25 Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly         30                  35                  40 Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn         45                  50                  55 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser         60                  65                  70 Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu         75                  80                  85 Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly         90                  95                  100 Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Trp Thr         105                 110                 115 Leu Glu Asn Ala Gln Ala Gln Gln         120                 125 DNA sequence #A9 pMON13215  116w ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA [SEQ ID NO:220] GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC TTCTATCTGT GGACCTTGGA GAACGCGCAG GCTCAACAG

EXAMPLE 65

Construction of pMON13252

Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Asp⁽ ⁵⁰⁾ variant, from Table 6, was digested with NcoI and HindIII resulting in a 345 base pair NcoI/HindIII fragment. This 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Plasmid DNA was isolated and screened by restriction analysis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated pMON13252. The plasmid, pMON13252, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

PEPTIDE A10; (15-125)HIL-3 ASP⁽⁵⁰⁾ pMON13252

        Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu [SEQ ID NO:218]         15                  20                  25 Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly         30                  35                  40 Glu Asp Gln Asp Ile Leu Met Asp Asn Asn Leu Arg Arg Pro Asn         45                  50                  55 Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser         60                  65                  70 Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu         75                  80                  85 Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly         90                  95                  100 Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr         105                 110                 115 Leu Glu Asn Ala Gln Ala Gln Gln         120                 125 DNA sequence #A10 pMON13252  50D ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA [SEQ ID NO:216] GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG

TABLE 7 position/ mutant NATIVE SUBSTITUTION RELATIVE pMON number (15-125) hiL-3 amino acid amino acid SEQ ID NO: POTENCY pMON13201 45/31M Gln Met 19 6.3 pMON13202 51/37R Asn Arg 19 1.58 pMON13203 51/37P Asn Pro 19 2.5 pMON13204 51/37T Asn Thr 19 3.16 pMON13205 56/42S Pro Ser 19 6.3 pMON13206 98/84I His Ile 19 6.3 pMON13207 45/31V Gln Val 19 4 pMON13208 42/28D Gly Asp 19 6.3 pMON13209 42/28S Gly Ser 19 12.6 pMON13210 42/28A Gly Ala 19 2.5 pMON13211 46/32S Asp Ser 19 16 pMON13212 82/68W Leu Trp 19 5 pMON13213 82/68D Leu Asp 19 4 pMON13214 100/86R Lys Arg 19 4 pMON13215 116/102W Lys Trp 19 31 pMON13216 23/9L Ile Leu 19 4 pMON13217 32/18R Leu Arg 19 7.9 pMON13218 32/18N Leu Asn 19 2 pMON13219 32/18A Leu Ala 19 1.58 pMON13220 34/20S Leu Ser 19 6.3 pMON13221 34/20M Leu Met 19 6.3 pMON13222 50/36D Glu Asp 19 7.9 pMON13223 62/48I Asn Ile 19 * pMON13224 166/52R Lys Arg 19 4 pMON13225 176/62P Ser Pro 19 1.25 pMON13226 77/63L Ile Leu 19 1.58 pMON13227 22/8G Glu Gly 19 0.008 pMON13228 115/101M Leu Met 19 0.04 pMON13229 122/108I Gln Ile 19 1 pMON13231 51/37H Asn His 19 1.25 pMON13232 59/45L Glu Leu 19 1.99 pMON13233 63/49H Arg His 19 * pMON13234 64/50N Ala Asn 19 0.03 pMON13235 65/51T Val Thr 19 1.58 pMON13236 76/62V Ser Val 19 2.5 pMON13237 76/62A Ser Ala 19 5 pMON13238 91/77P Ala Pro 19 * pMON13240 100/86Q Lys Gln 19 2.5 pMON13241 101/87M Asp Met 19 6.3 pMON13242 105/91N Asn Asn 19 * pMON13243 116/102V Lys Val 19 79 pMON13244 122/108F Gln Phe 19 6.3 pMON13245 123/109E Ala Gln 19 1.58 position/ mutant NATIVE SUBSTITUTION RELATVE pMON number (1-133) hiL-3 amino acid amino acid SEQ ID NO: POTENCY pMON13246 42D Gly Asp 15 20 pMON13247 42S Gly Ser 15 * pMON13248 42A Gly Ala 15 16 pMON13249 45V Gln Val 15 5 pMON13250 45M Gln Met 15 * pMON13251 46S Asp Ser 15 5 pMON13252 50D Glu Asp 15 5 pMON13253 981 His Ile 15 * pMON13264 97V Ile Val 15 4 pMON13266 75K Glu Lys 15 0.25 pMON13267 89N Thr Asn 15 2.5

Table 7 shows the biological activity of (15-125) hIL-3 mutant polypeptides of the present invention expressed from Intracellular expression vectors. Upon expression these muteins may have Met- or Met-Ala-preceding the initial (15-125) hIL-3 amino acid. The relative biological activity of IL-3 mutants is calculated by dividing the EC₅₀ (1-133) hIL-3 by the EC₅₀ of the mutant.

EXAMPLE 66

The variants in Table 8 were constructed by cassette mutagenesis using methods described in the Materials and Methods and the Examples contained herein, particularly Examples 54-57. Parental plasmid DNA (Table 8), digested with the appropriate restriction enzymes (Table 8), was ligated with the indicated annealed pairs of complementary oligonucleotides (Table 8). The assembled oligonucleotides create appropriate restriction ends and a portion of the (15-125) hIL-3 gene sequence Individual isolates were screened by restriction analysis and DNA sequenced to confirm that the desired changes in the (15-125) hIL-3 variant gene were made. The oligonucleotides create change(s) in the (15-125) hIL-3 gene which enclode the corresponding amino acid substitution in the variant polypeptide (Table 8). The amino acids substitutions in polypeptide #1 (SEQ ID NO:65) are indicated in Table 8.

position/ sub- restriction native a.a. stitution codon oligo pair oligo pair oligo pair oligo pair oligo pair parental plasmid digest 21 asp glu GAA 21glu1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:73 SEQ ID NO:523 SEQ ID NO:524 21glu4 NcoRV5 NcoRV6 SEQ ID NO:219 SEQ ID NO:526 SEQ ID NO:527 21 asp gln CAA 21gln1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:71 SEQ ID NO:523 SEQ ID NO:524 21gln4 NcoRV5 NcoRV6 SEQ ID NO:72 SEQ ID NO:526 SEQ ID NO:527 21 asp asn AAC 21asn1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:68 SEQ ID NO:523 SEQ ID NO:524 21asn4 NcoRV5 NcoRV6 SEQ ID NO:70 SEQ ID NO:526 SEQ ID NO:527 21 asp thr ACC 21thr1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:232 SEQ ID NO:523 SEQ ID NO:524 21thr4 NcoRV5 NcoRV6 SEQ ID NO:233 SEQ ID NO:526 SEQ ID NO:527 21 asp ser AGC 21ser1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:230 SEQ ID NO:523 SEQ ID NO:524 21ser4 NcoRV5 NcoRV6 SEQ ID NO:231 SEQ ID NO:526 SEQ ID NO:527 22 glu asp GAC 22asp1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:236 SEQ ID NO:523 SEQ ID NO:524 22asp4 NcoRV5 NcoRV6 SEQ ID NO:237 SEQ ID NO:526 SEQ ID NO:527 22 glu asn AAC 22asn1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:234 SEQ ID NO:523 SEQ ID NO:524 22asn4 NcoRV5 NcoRV6 SEQ ID NO:235 SEQ ID NO:526 SEQ ID NO:527 22 glu gln CAG 22gln1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:238 SEQ ID NO:523 SEQ ID NO:524 22gln4 NcoRV5 NcoRV6 SEQ ID NO:239 SEQ ID NO:526 SEQ ID NO:521 22 glu leu CTG 22leu1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:240 SEQ ID NO:523 SEQ ID NO:524 22leu4 NcoRV5 NcoRV6 SEQ ID NO:241 SEQ ID NO:526 SEQ ID NO:527 22 glu val GTT 22val1 NcoRv2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:242 SEQ ID NO:523 SEQ ID NO:524 22val4 NcoRV5 NcoRV6 SEQ ID NO:243 SEQ ID NO:526 SEQ ID NO:527 34 leu glu GAA NcoRV1 34Glu2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:251 SEQ ID NO:524 NcoRV4 34Glu5 NcoRV6 SEQ ID NO:525 SEQ ID NO:252 SEQ ID NO:527 34 leu gln GAG NcoRV1 34gln2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:248 SEQ ID NO:524 NcoRV4 34gln5 NcoRV6 SEQ ID NO:525 SEQ ID NO:249 SEQ ID NO:527 34 leu thr ACC NcoRV1 34thr2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:256 SEQ ID NO:524 NcoRV4 34thr5 NcoRV6 SEQ ID NO:525 SEQ ID NO:257 SEQ ID NO:527 34 leu arg CGT NcoRV1 34arg2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:246 SEQ ID NO:524 NcoRV4 34arg5 NcoRV6 SEQ ID NO:525 SEQ ID NO:247 SEQ ID NO:527 34 leu ala GCT NcoRV1 34ala2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:244 SEQ ID NO:524 NcoRV4 34ala5 NcoRV6 SEQ ID NO:525 SEQ ID NO:245 SEQ ID NO:527 34 leu phe TTC NcoRV1 34phe2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:254 SEQ ID NO:524 NcoRV4 34phe5 NcoRV6 SEQ ID NO:525 SEQ ID NO:255 SEQ ID NO:527 34 leu ile ATC NcoRV1 34ile2 NcoRV3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:252 SEQ ID NO:524 NcoRV4 34ile5 NcoRV6 SEQ ID NO:525 SEQ ID NO:253 SEQ ID NO:527 42 gly lys AAA NcoRV1 NcoRv2 42lys3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:268 NcoRV4 NcoRV5 42lys6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:269 42 gly asn AAC NcoRV1 NcoRv2 42asn3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:260 NcoRV4 NcoRV5 42asn6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:261 42 gly thr ACC NcoRV1 NcoRv2 42thr3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:274 NcoRV4 NcoRV5 42thr6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:275 42 gly leu CTG NcoRV1 NcoRv2 42leu3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:266 NcoRV4 NcoRV5 42leu6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:267 42 gly val GTT NcoRV1 NcoRv2 42val3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:278 NcoRV4 NcoRV5 42val6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:279 42 gly glu GAA NcoRV1 NcoRv2 42glu3 PMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:262 NcoRV4 NcoRV5 42glu6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:263 42 gly phe TTC NcoRV1 NcoRv2 42phe3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:272 NcoRV4 NcoRV5 42phe6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:273 42 gly tyr TAC NcoRV1 NcoRv2 42tyr3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:276 NcoRV4 NcoRV5 42tyr6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:277 42 gly ile ATC NcoRV1 NcoRv2 42ile3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:264 NcoRV4 NcoRV5 42ile6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:265 42 gly met ATG NcoRV1 NcoRv2 42met3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:270 NcoRV4 NcoRV5 42met6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:271 43 glu gln CAG NcoRV1 NcoRv2 43gln3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:282 NcoRV4 NcoRV5 43gln6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:283 43 glu arg CGT NcoRV1 NcoRv2 43arg3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:280 NcoRV4 NcoRV5 43arg6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:281 43 glu thr ACC NcoRV1 NcoRv2 43thr3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:286 NcoRV4 NcoRV5 43thr6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:287 43 glu gly GGT NcoRV1 NcoRv2 43gly3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:284 NcoRV4 NcoRV5 43gly6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:285 44 asp glu GAA NcoRV1 NcoRv2 44glu3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:294 NcoRV4 NcoRV5 44glu6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:295 44 asp asn AAC NcoRV1 NcoRv2 44asn3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:290 NcoRV4 NcoRV5 44asn6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:291 44 asp gln CAG NcoRV1 NcoRv2 44gln3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:292 NcoRV4 NcoRV5 44gln6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:293 44 asp ala GCT NcoRV1 NcoRv2 44ala3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:288 NcoRV4 NcoRV5 44ala6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:289 45 gln asp GAC NcoRV1 NcoRv2 45asp3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:302 NcoRV4 NcoRV5 45asp6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:303 45 gln asn AAC NcoRV1 NcoRv2 45asn3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:300 NcoRV4 NcoRV5 45asn6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:301 45 gln arg CGT NcoRV1 NcoRv2 45arg3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:298 NcoRV4 NcoRV5 45arg6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:299 45 gln ser TCC NcoRV1 NcoRv2 45ser3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:310 NcoRV4 NcoRV5 45ser6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:311 45 gln ala GCT NcoRV1 NcoRv2 45ala3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:296 NcoRV4 NcoRV5 45ala6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:297 45 gln ile ATC NcoRV1 NcoRv2 45ile3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:308 NcoRV4 NcoRV5 45ile6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:309 45 gln glu GAA NcoRV1 NcoRv2 45glu3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:304 NcoRV4 NcoRV5 45glu6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:305 45 gln his CAC NcoRV1 NcoRv2 45his3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:306 NcoRV4 NcoRV5 45his6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:307 46 asp glu GAA NcoRV1 NcoRv2 46glu3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:318 NcoRV4 NcoRV5 46glu6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:319 46 asp asn AAC NcoRV1 NcoRv2 46asn3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:314 NcoRV4 NcoRV5 46asn6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:315 46 asp gln CAG NcoRV1 NcoRv2 46gln3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:316 NcoRV4 NcoRV5 46gln6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:317 46 asp lys AAA NcoRV1 NcoRv2 46lys3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:326 NcoRV4 NcoRV5 46lys6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:327 46 asp his CAC NcoRV1 NcoRv2 48his3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:320 NcoRV4 NcoRV5 46his6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:321 46 asp ala GCT NcoRV1 NcoRv2 48ala3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:312 NCORV4 NcoRV5 48ala6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:313 46 asp tyr TAC NcoRV1 NcoRv2 46tyr3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:328 NcoRV4 NcoRV5 46tyr6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:329 46 asp ile ATC NcoRV1 NcoRv2 46ile3 pMON13356 Ncol, EcoRV SEQ ID NO: 522 SEQ ID NO:523 SEQ ID NO:322 NcoRV4 NcoRV5 46ile6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:323 46 asp val GTT NcoRV1 NcoRV2 46val3 pMON13356 Ncol, EcoRV SEQ ID NO:522 SEQ ID NO:523 SEQ ID NO:330 NcoRV4 NcoRV5 46val6 SEQ ID NO:525 SEQ ID NO:526 SEQ ID NO:331 48 leu glu GAA 48glu1 RVNsl2 RVNsl3 pMON13367 EcoRV, Ncol SEQ ID NO:334 SEQ ID NO:528 SEQ ID NO:530 48glu4 RVNsl5 RVNsl6 SEQ ID NO:535 SEQ ID NO:532 SEQ ID NO:533 48 leu lys AAA 48lys1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:336 SEQ ID NO:529 SEQ ID NO:530 48lys4 RVNsl5 RVNSl6 SEQ ID NO:337 SEQ ID NO:532 SEQ ID NO:529 48 leu thr ACC 48thr1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:340 SEQ ID NO:525 SEQ ID NO:530 48thr4 RVNsl5 RVNsl5 SEQ ID NO:341 SEQ ID NO:532 SEQ ID NO:533 48 leu ala GCT 48ala1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:332 SEQ ID NO:529 SEQ ID NO:530 48ala4 RVNsl5 RVNsl6 SEQ ID NO:333 SEQ ID NO:532 SEQ ID NO:533 48 leu met ATG 48met1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:338 SEQ ID NO:529 SEQ ID NO:530 48met4 RVNsl5 RVNsl6 SEQ ID NO:339 SEQ ID NO:532 SEQ ID NO:533 48 leu val CAC 48val1 RVNsl2 RVNs13 pMON13357 EcoRV, Ncol SEQ ID NO:342 SEQ ID NO:529 SEQ ID NO:530 48val4 RVNsl5 RVNsl6 SEQ ID NO:343 SEQ ID NO:532 SEQ ID NO:533 50 glu lys AAA 50lys1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:356 SEQ ID NO:529 SEQ ID NO:530 501ys4 RVNsl5 RVNsl5 SEQ ID NO:357 SEQ ID NO:532 SEQ ID NO:533 50 glu asn AAC 50asn1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:352 SEQ ID NO:529 SEQ ID NO:530 50asn4 RVNsl5 RVNsl6 SEQ ID NO:353 SEQ ID NO:532 SEQ ID NO:533 50 glu ser TCC 50ser1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:358 SEQ ID NO:529 SEQ ID NO:530 50ser4 RVNsl5 RVNsl6 SEQ ID NO:359 SEQ ID NO:532 SEQ ID NO:533 50 glu ala GCT 50ala1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:350 SEQ ID NO:529 SEQ ID NO:530 50ala4 RVNsl5 RVNsl6 SEQ ID NO:351 SEQ ID NO:532 SEQ ID NO:533 50 glu ile ATC 50ile1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:354 SEQ ID NO:529 SEQ ID NO:530 50ile4 RVNsl5 RVNsl6 SEQ ID NO:355 SEQ ID NO:532 SEQ ID NO:533 50 glu val GTT 50val1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:360 SEQ ID NO:529 SEQ ID NO:530 50val4 RVNsl5 RVNsl6 SEQ ID NO:361 SEQ ID NO:532 SEQ ID NO:533 50 glu his CAC 50his1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:344 SEQ ID NO:529 SEQ ID NO:530 50his4 RVNsl5 RVNsl6 SEQ ID NO:345 SEQ ID NO:532 SEQ ID NO:533 50 glu phe TTC 50phe1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:348 SEQ ID NO:529 SEQ ID NO:530 50phe4 RVNsl5 RVNsl6 SEQ ID NO:349 SEQ ID NO:532 SEQ ID NO:533 50 glu met ATG 50met1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:346 SEQ ID NO:529 SEQ ID NO:530 50met4 RVNsl5 RVNsl6 SEQ ID NO:347 SEQ ID NO:532 SEQ ID NO:533 54 arg asn AAC 54asn1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:364 SEQ ID NO:529 SEQ ID NO:530 54asn4 RVNsl5 RVNsl6 SEQ ID NO:365 SEQ ID NO:532 SEQ ID NO:533 54 arg lys AAA 54lys1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:368 SEQ ID NO:529 SEQ ID NO:530 54lys4 RVNsl5 RVNsl6 SEQ ID NO:369 SEQ ID NO:532 SEQ ID NO:533 54 arg his CAC 54his1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:366 SEQ ID NO:529 SEQ ID NO:53O 54his4 RVNsl5 RVNsl6 SEQ ID NO:367 SEQ ID NO:532 SEQ ID NO:533 54 arg ala GCT 54ala1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:362 SEQ ID NO:529 SEQ ID NO:530 54ala4 RVNsl5 RVNsl6 SEQ ID NO:363 SEQ ID NO:532 SEQ ID NO:533 56 pro glu GAA 56glu1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:376 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56glu5 RVNsl6 SEQ ID NO:531 SEQ ID NO:377 SEQ ID NO:533 56 pro gln 56gln1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:314 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56gln5 RVNsl6 SEQ ID NO:531 SEQ ID NO:375 SEQ ID NO:533 58 pro arg CGT 56arg1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:372 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56arg5 RVNsl6 SEQ ID NO:531 SEQ ID NO:373 SEQ ID NO:533 56 pro his CAC 56his1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:378 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56his5 RVNsl6 SEQ ID NO:531 SEQ ID NO:379 SEQ ID NO:533 56 pro thr ACC 56thr1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:384 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56thr5 RVNsl6 SEQ ID NO:531 SEQ ID NO:385 SEQ ID NO:533 56 pro ala GCT 56ala1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:370 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56ala5 RVNsl6 SEQ ID NO:531 SEQ ID NO:371 SEQ ID NO:533 56 pro tyr TAC 56tyr1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:386 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56tyr5 RVNsl6 SEQ ID NO:531 SEQ ID NO:387 SEQ ID NO:533 56 pro phe TTC 56phe1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:382 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56phe5 RVNsl6 SEQ ID NO:531 SEQ ID NO:383 SEQ ID NO:533 56 pro leu CTG 56leu1 RVNsl2 RVNsl3 pMON133S1 EcoRV, Ncol SEQ ID NO:380 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56leu5 RVNsl6 SEQ ID NO:531 SEQ ID NO:381 SEQ ID NO:533 56 pro val GTT 56val1 RVNsl2 RVNsl3 pMON13357 EcoRV, Ncol SEQ ID NO:388 SEQ ID NO:529 SEQ ID NO:530 RVNsl4 56val5 RVNsl6 SEQ ID NO:531 SEQ ID NO:389 SEQ ID NO:533 82 leu glu GAA NslEco1 82glu2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:94 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82glu6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:395 SEQ ID NO:542 SEQ ID NO:545 82 leu asn AAC NslEcol 82asn2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:392 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82asn6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:393 SEQ ID NO:542 SEQ ID NO:545 82 leu his CAC NslEco1 82his2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:396 SEQ ID NO:538 SEQ ID NO:539 NslEco5 82his6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:397 SEQ ID NO:542 SEQ ID NO:545 82 leu thr ACC NslEco1 82thr2 NslEco3 NslEc04 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:406 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82thr6 NslEco7 NslEco3 SEQ ID NO:540 SEQ ID NO:407 SEQ ID NO:542 SEQ ID NO:545 82 leu ser TCC NslEco1 82ser2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:404 SEQ ID NO:538 SEQ ID NO:539 NslEco5 82ser6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:405 SEQ ID NO:542 SEQ ID NO:545 82 leu ala GCT NslEco1 82ala2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:390 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82ala6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:391 SEQ ID NO:542 SEQ ID NO:545 82 leu tyr TAC NslEco1 82tyr2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:408 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82tyr6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:409 SEQ ID NO:542 SEQ ID NO:545 82 leu phe TTC NslEco1 82phe2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:402 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82phe6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:403 SEQ ID NO:542 SEQ ID NO:545 82 leu ile ATC NslEco1 82ile2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:398 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82ile6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:399 SEQ ID NO:542 SEQ ID NO:545 82 leu met ATG NslEco1 82met2 NslEco3 NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:400 SEQ ID NO:536 SEQ ID NO:539 NslEco5 82met6 NslEco7 NslEco8 SEQ ID NO:540 SEQ ID NO:401 SEQ ID NO:542 SEQ ID NO:545 92 pro ala GCT NslEco1 NslEco2 92ala3A NslEco3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:410 SEQ ID NO:538 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 92ala7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:411 SEQ ID NO:545 92 pro gly GGT NslEco1 NslEco2 92gly3A NslEco3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:412 SEQ ID No:538 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 92ala7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:413 SEQ ID NO:545 92 pro ile ATC NslEco1 NslEco2 92ile3A NslEco3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:414 SEQ ID NO:538 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 92ala7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:415 SEQ ID NO:545 94 arg gln GAG NslEco1 NslEco2 NslEco3A 94gln3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:418 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 94gln7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:419 SEQ ID NO:545 94 arg lys AAA NslEco1 NslEco2 NslEco3A 94lys3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:422 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 94lys7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:423 SEQ ID NO:545 94 arg his CAC NslEco1 NslEco2 NslEco3A 94his3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:420 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 94his7B NslEco8 SEQ ID NO:540 SEQ ID NO:538 SEQ ID NO:543 SEQ ID NO:421 SEQ ID NO:545 94 arg ala GCT NslEco1 NslEco2 NslEco3A 94ala3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:416 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 94ala7B NslEco8 SEQ ED NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:417 SEQ ID NO:545 95 his asn AAC NslEco1 NslEco2 NslEco3A 95asn3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:426 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 95asn7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:427 SEQ ID NO:545 95 his lys AAA NslEco1 NslEco2 NslEco3A 95lys3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:432 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 95lys7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:433 SEQ ID NO:545 95 his ser TCC NslEco1 NslEco2 NslEco3A 95ser3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:438 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 95ser7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:439 SEQ ID NO:545 95 his ala GCT NslEco1 NslEco2 NslEco3A 95ala3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:424 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 95ala7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:425 SEQ ID NO:545 95 his trp TGG NslEco1 NslEco2 NslEco3A 95trp3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:440 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 95trp7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:441 SEQ ID NO:545 95 his phe TTC NslEco1 NslEco2 NslEco3A 95phe3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO11 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 95phe7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:437 SEQ ID NO:545 95 his ile ATC NslEco1 NslEco2 NslEco3A 95ile3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:430 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 95ile7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID No:431 SEQ ID NO:545 98 his glu GAA NslEco1 NslEco2 NslEco3A 98glu3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID No:446 SEQ ID NO:539 NslEco5 NslEco5 NslEco7A 98glu7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID No:447 SEQ ID NO:545 98 his gln CAA NslEco1 NslEco2 NslEco3A 98gln3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID No:444 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98gln7B NslEco8 SEQ ID NQ:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:445 SEQ ID NO:545 98 his ser TCC NslEco1 NslEco2 NslEco3A 98ser3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID No:452 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98ser7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID No:453 SEQ ID NO:545 98 his phe TTC NslEco1 NslEco2 NslEco3A 98phe3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:450 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98phe7B NslEco3 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:451 SEQ ID NO:545 98 his met ATG NslEco1 NslEco2 NslEco3A 98met3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:446 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98met7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:449 SEQ ID NO:545 98 his val GTA NslEco1 NslEco2 NslEco3A 98val3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:454 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98val7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:455 SEQ ID NO:545 98 his lys AAA NslEco1 NslEco2 NslEco3A 98lys3B NsilEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:458 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98lys7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:459 SEQ ID NO:545 98 his arg CGT NslEco1 NslEco2 NslEco3A 98arg3B NslEco4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:456 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98arg7B NslEco8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:457 SEQ ID NO:545 98 his tyr TAC NslEco1 NslEco2 NslEco3A 98tyr3B NslEc04 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:537 SEQ ID NO:460 SEQ ID NO:539 NslEco5 NslEco6 NslEco7A 98tyr7B NslEco5 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:543 SEQ ID NO:461 SEQ ID NO:545 101 asp glu GAA NslEco1 NslEco2 NslEco3 101glu4 pMON13358 Nsil, EcoRI SEQ ED NO:534 SEQ ID NO:535 SEQ ID NO:536 SEQ ID NO:466 NslEco5 NslEco6 NslEco7 101glu8 SEQ ED NO:540 SEQ ID NO:541 SEQ ID NO:542 SEQ ID NO:467 101 asp asn AAC NslEco1 NslEco2 NslEco3 101asn4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:536 SEQ ID NO:464 NslEco5 NslEco6 NslEco7 101asn8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:542 SEQ ID NO:465 101 asp ser TTC NslEco1 NslEco2 NslEco3 101ser4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:536 SEQ ID NO:476 NslEco5 NslEco6 NslEco7 101ser8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:542 SEQ ID NO:477 101 asp ala GCT NslEco1 NslEco2 NslEco3 101ala4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:536 SEQ ID NO:462 NslEco5 NslEco6 NslEco7 101ala8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:542 SEQ ID NO:463 101 asp gly GGT NslEco1 NslEco2 NslEco3 101gly4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:335 SEQ ID NO:536 SEQ ID NO:466 NslEco5 NslEco6 NslEco7 101gly8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:542 SEQ ID NO:469 101 asp ile ATC NslEco1 NslEco2 NslEco3 101ile4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:536 SEQ ID NO:470 NslEco5 NslEco6 NslEco7 101ile8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:542 SEQ ID NO:471 101 asp leu CTG NslEco1 NslEco2 NslEco3 101leu4 pMON13358 Nsil, EcoRI SEQ ID NO:534 SEQ ID NO:535 SEQ ID NO:536 SEQ ID NO:472 NslEco5 NslEco6 NslEco7 101leu8 SEQ ID NO:540 SEQ ID NO:541 SEQ ID NO:542 SEQ ID NO:473 108 arg gln CAG 108gln1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:480 SEQ ID NO:547 108gln3 EcoHin4 SEQ ID NO:481 SEQ ID NO:549 108 arg his CAC 108his1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:482 SEQ ID NO:547 108his3 EcoHin4 SEQ ID NO:483 SEQ ID NO:549 108 arg ser TCC 108ser1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:484 SEQ ID NO:547 108ser3 EcoHin4 SEQ ID NO:485 SEQ ID NO:549 108 arg ala GCT 108ala1 EcoHin2 pMON13359 EcoRi, HinDIII SEQ ID NO:478 SEQ ID NO:547 108ala3 EcoHin4 SEQ ID NO:479 SEQ ID NO:549 110 lys arg CGT 110arg1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:46 SEQ ID NO:547 110arg3 EcoHin4 SEQ ID NO:487 SEQ ID NO:549 110 lys his CAC 110his1 EcoHin2 pMON13359 EcoRI, HinDILL SEQ ID NO:490 SEQ ID NO:547 110his3 EcoHin4 SEQ ID NO:491 SEQ ID NO:549 110 lys glu GAA 110glu1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:488 SEQ ID NO:547 110glu3 EcoHin4 SEQ ID NO:499 SEQ ID NO:549 110 lys ser TCC 110ser1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:494 SEQ ID NO:547 110ser3 EcoHin4 SEQ ID NO:495 SEQ ID NO:549 110 lys ala GCT 110ala1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:492 SEQ ID NO:547 110la3 EcoHin4 SEQ ID NO:493 SEQ ID NO:549 113 phe asp GAC 113asp1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:496 SEQ ID NO:547 113asp3 EcoHin4 SEQ ID NO:497 SEQ ID NO:549 113 phe lys AAA 113lys1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:502 SEQ ID NO:547 113lys3 EcoHin4 SEQ ID NO:503 SEQ ID NO:549 113 phe leu CTG 113leu1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:500 SEQ ID NO:547 113leu3 EcoHin4 SEQ ID NO:501 SEQ ID NO:549 113 phe ile ATC 113ile1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:498 SEQ ID NO:541 113ile3 EcoHin4 SEQ ID NO:499 SEQ ID NO:549 113 phe val GTT 113val1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:504 SEQ ID NO:547 113va13 EcoHin4 SEQ ID NO:505 SEQ ID NO:549 116 lys asn AAC 116asn1 EcoHin2 pMONI3359 EcoRI, HinDIII SEQ ID NO:510 SEQ ID NO:547 116asn3 EcoHin4 SEQ ID NO:511 SEQ ID NO:549 116 lys arg CGT 116arg1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:506 SEQ ID NO:547 116arg3 EcoHin4 SEQ ID NO:509 SEQ ID NO:549 116 lys his CAC 116his1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:514 SEQ ID NO:547 116his3 EcoHin4 SEQ ID NO:515 SEQ ID NO:549 116 lys ala GCT 116ala1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:506 SEQ ID NO:547 116ala3 EcoHin4 SEQ ID NO:507 SEQ ID NO:549 116 lys tyr TAC 116tyr1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:520 SEQ ID NO:547 116tyr3 EcoHin4 SEQ ID NO:521 SEQ ID NO:549 116 lys phe TTC 116phe1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:518 SEQ ID NO:547 116phe3 EcoHin4 SEQ ID NO:519 SEQ ID NO:549 116 lys gln CAG 116gln1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:512 SEQ ID NO:547 116gln3 EcoHin4 SEQ ID NO:513 SEQ ID NO:549 116 lys met ATG 116met1 EcoHin2 pMON13359 EcoRI, HinDIII SEQ ID NO:516 SEQ ID NO:547 116met3 EcoHin4 SEQ ID NO:517 SEQ ID NO:549

It will be apparent to those skilled in the art that other codons besides those shown in Table 8 can also code for the substituted amino acids in the hIL-3 muteins. The present invention includes the DNAs encoding the mutant hIL-3 polypeptides of the invention including the various codons which can code for the parental and substituted amino acids of the hIL-3 muteins of the invention due to the degeneracy of the genetic code.

hIL-3 (15-125) variant genes encoding the variants listed in Table 8 can also be expressed from intracellular expression vectors to produce large quantities of the variant protein which can be purified and assayed for biological activity. The hIL-3 variant genes, from Table 8, can be excised from the secretion expression vector, as a 345 base pair NcoI/HindIII fragment and ligated into an appropriate intrecellular expression vector, such as pMON2341 digested with NcoI and HindIII.

Table 9 shows the biological activity of (15-125) hIL-3 muteins of the present invention which have one amino acid substitutions in the (15-125) hIL-3 polypeptide and which were constructed as described in Example 66. The mutants in Table 9 were secreted into the periplasmic space in E. coli. The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (1-125) hIL-3 (pMON6458 or pMMON5988). The relative biological activity of IL-3 mutants is calculated by dividing the EC₅₀ (1-133) hIL-3 by the EC₅₀ of the mutant. The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “*”0 indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

TABLE 9 (15-125) hIL-3 MUTANT PARENTAL BIOL aa position AA codon AA SEQ ID NO: codon ACTIVITY 21/7 ASP GAT ASN 19 AAC 0.01 21/7 ASP GAT GLN 19 CAA 0.07 21/7 ASP GAT GLU 19 GAA 0.5 21/7 ASP GAT SER 19 AGC 0.1 21/7 ASP GAT THR 19 ACC 0.1 22/8 GLU GAA ASN 19 AAC * 22/8 GLU GAA ASP 19 GAC * 22/8 GLU GAA GLN 19 CAG <0.01 22/8 GLU GAA LEU 19 CTG * 22/8 GLU GAA VAL 19 GTT * 34/20 LEU TTG ALA 19 GCT 2.2 34/20 LEU TTG ARG 19 CGT 2.2 34/20 LEU TTG GLN 19 CAG 1.1 34/20 LEU TTG GLU 19 GAA 1.5 34/20 LEU TTG ILE 19 ATC 1.3 34/20 LEU TTG PHE 19 TTC 1.8 34/20 LEU TTG THR 19 ACC 1.1 42/28 GLY GGG ASN 19 AAC 1.3 (3) 0.28 42/28 GLY GGG ILE 19 ATC 10 42/28 GLY GGG LEU 19 CTG 10.1 (3) 7.57 42/28 GLY GGG MET 19 ATG 2.2 (3) 1.14 42/28 GLY GGG TYR 19 TAC 11 (2) 8.9 42/28 GLY GGG VAL 19 GTT 0.33 43/29 GLU GAA ARG 19 CGT * 43/29 GLU GAA GLN 19 CAG <0.004 43/29 GLU GAA GLY 19 GGT * 43/29 GLU GAA THR 19 ACC 0.005 44/30 ASP GAC ALA 19 GCT * 44/30 ASP GAC ASN 19 AAC * 44/30 ASP GAC GLN 19 CAG * 44/30 ASP GAC GLU 19 GAA 0.66 45/31 GLN CAA ALA 19 GCT 1 45/31 GLN CAA ASN 19 AAC 15.8 45/31 GLN CAA GLU 19 GAA 2.3 45/31 GLN CAA ILE 19 ATC 4.9 45/31 GLN CAA SER 19 TCC 0.7 46/32 ASP GAC ALA 19 GCT 6.3 46/32 ASP GAC ASN 19 AAC 0.66, 1.1 46/32 ASP GAC GLN 19 CAG 6.3 46/32 ASP GAC GLU 19 GAA 1.97 (3) 2.14 46/32 ASP GAC HIS 19 CAC 3.2, 1.4 46/32 ASP GAC ILE 19 ATC 0.5 46/32 ASP GAC LYS 19 AAA 0.5 46/32 ASP GAC TYR 19 TAC 0.66 46/32 ASP GAC VAL 19 GTT 6.3 48/34 LEU CTG GLU 19 GAA * 48/34 LEU CTG HIS 19 * 48/34 LEU CTG LYS 19 AAA * 48/34 LEU CTG THR 19 ACC * 48/34 LEU CTG VAL 19 CAC * 50/36 GLU GAA ALA 19 GCT 0.5 50/36 GLU GAA ASN 19 AAC 1.7 50/36 GLU GAA HIS 19 CAC * 50/36 GLU GAA LYS 19 AAA * 50/36 GLU GAA SER 19 TCC 1.3 50/36 GLU GAA VAL 19 GTT * 54/40 ARG CGA ALA 19 GCT 0.9 54/40 AFG CGA ASN 19 AAC * 54/40 ARG CGA HIS 19 CAC 0.01 54/40 ARG CGA LYS 19 AAA 0.2 56/42 PRO CAA ALA 19 GCT 1.8 56/42 PRO CAA ASN 19 0.6 56/42 PRO CAA ARG 19 CGT 1.2 56/42 PRO CAA GLU 19 GAA 0.9 56/42 PRO CAA HIS 19 CAC 0.4 56/42 PRO CAA LEU 19 CTG 1.2 56/42 PRO CAA PHE 19 TTC 56/42 PRO CAA THR 19 ACC 0.6 56/42 PRO CAA VAL 19 GTT 1.1 82/68 LEU CTG ALA 19 GCT 0.5 82/68 LEU CTG ASN 19 AAC 2.9 82/68 LEU CTG GLU 19 GAA 4.57 (3) 5.0 82/68 LEU CTG HIS 19 CAC 2.2 82/68 LEU CTG ILE 19 ATC 0.8 82/68 LEU CTG MET 19 ATG 1.1 82/68 LEU CTG PHE 19 TTC 3.2 82/68 LEU CTG SER 19 TCC 2.2 82/68 LEU CTG THR 19 ACC 1.6 82/68 LEU CTG TYR 19 TAC 2.7 94/80 ARG CGA GLN 19 CAG 0.03 94/80 ARG CGA HIS 19 CAC 0.01 94/80 ARG CGA LYS 19 AAA * 95/81 HIS CAT ASN 19 AAC 2.7 (2) 2.3 95/81 HIS CAT ILE 19 ATC 0.33 95/81 HIS CAT LYS 19 AAA 0.9 95/81 HIS CAT MET 19 ATG 1 95/81 HIS CAT PHE 19 TTC 0.66 95/81 HIS CAT SER 19 TCC 4 95/81 HIS CAT TRP 19 TGG * 98/84 HIS CAT ARG 19 CGT 3.2 98/84 HIS CAT GLN 19 CAA 2.2 98/84 HIS CAT GLU 19 GAA 1.55 (2) 0.15 98/84 HIS CAT LYS 19 AAA 4 98/84 HIS CAT MET 19 ATG 2.2 98/84 HIS CAT PHE 19 TTC 1 98/84 HIS CAT SER 19 TCC 4 98/84 HIS CAT THR 19 2.2 98/84 HIS CAT VAL 19 GTA 2.4 (2) 0.8 101/87 ASP GAC ASN 19 AAC 7 101/87 ASP GAC GLU 19 GAA * 101/87 ASP GAC ILE 19 ATC 3.2 101/87 ASP GAC LEU 19 CTG 3.2 108/94 ARG CGG ALA 19 GCT 4 108/94 ARG CGG GLN 19 CAG 0.4 108/94 ARG CGG HIS 19 CAC * 108/94 ARG CGG SER 19 TCC 3.7 110/96 LYS AAA GLU 19 GAA * 110/96 LYS AAA HIS 19 CAC * 110/96 LYS AAA ILE 19 ATC * 113/99 PHE TTC ASP 19 GAC * 113/99 PHE TTC ILE 19 ATC * 113/99 PHE TTC LEU 19 CTG * 113/99 PHE TTC LYS 19 AAA * 116/102 LYS AAA ALA 19 GCT 5 116/102 LYS AAA ARG 19 CGT 0.03 116/102 LYS AAA ASN 19 AAC 0.22 116/102 LYS AAA GLN 19 CAG 0.33 116/102 LYS AAA HIS 19 CAC 3.2 116/102 LYS AAA MET 19 ATG 0.9 116/102 LYS AAA PHE 19 TTC 2.5 116/102 LYS AAA TYR 19 TAC 5.4 (2) 0.3

549 23 base pairs nucleic acid single linear DNA (synthetic) 1 CTAGCGATCT TTTAATAAGC TTG 23 23 base pairs nucleic acid single linear DNA (synthetic) 2 GATCCAAGCT TATTAAAAGA TCG 23 69 base pairs nucleic acid single linear DNA (synthetic) 3 GGCAACAATT TCTACAAAAC ACTTGATACT GTATGAGCAT ACAGTATAAT TGCTTCAACA 60 GAACAGATC 69 67 base pairs nucleic acid single linear DNA (synthetic) 4 TGTTCTGTTG AAGCAATTAT ACTGTATGCT CATACAGTAT CAAGTGTTTT GTAGAAATTG 60 TTGCCGC 67 23 base pairs nucleic acid single linear DNA (synthetic) 5 CCATTGCTGC CGGCATCGTG GTC 23 46 base pairs nucleic acid single linear DNA (synthetic) 6 CATGGCTCCA ATGACTCAGA CTACTTCTCT TAAGACTTCT TGGGTT 46 42 base pairs nucleic acid single linear DNA (synthetic) 7 AACCCAAGAA GTCTTAAGAG AAGTAGTCTG AGTCATTGGA GC 42 64 base pairs nucleic acid single linear DNA (synthetic) 8 AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGT 60 AATA 64 64 base pairs nucleic acid single linear DNA (synthetic) 9 AGCTTATTAC TGTTGAGCCT GCGCGTTCTC CAAGGTTTTC AGATAGAAGG TCAGTTTACG 60 ACGG 64 126 amino acids amino acid linear peptide 10 Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn 1 5 10 15 Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro 20 25 30 Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile 35 40 45 Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg 50 55 60 Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys 65 70 75 80 Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His 85 90 95 Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu 100 105 110 Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 115 120 125 24 base pairs nucleic acid single linear DNA (synthetic) 11 CATGGCTAAC TGCTCTAACA TGAT 24 22 base pairs nucleic acid single linear DNA (synthetic) 12 CGATCATGTT AGAGCAGTTA GC 22 113 amino acids amino acid linear peptide 13 Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys 1 5 10 15 Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp 20 25 30 Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala 35 40 45 Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser 50 55 60 Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro 65 70 75 80 Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg 85 90 95 Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln 100 105 110 Gln 27 amino acids amino acid linear peptide 14 Met Met Ile Thr Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala 1 5 10 15 Gly Val Met Ser Ala Gln Ala Met Ala Asn Cys 20 25 133 amino acids amino acid linear peptide Modified-site /note= “Met- may or may not precede the amino acid in position 1” Modified-site 17 /note= “Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg” Modified-site 18 /note= “Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln” Modified-site 19 /note= “Xaa at positiion 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys” Modified-site 20 /note= “Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala” Modified-site 21 /note= “Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser, or Val” Modified-site 22 /note= “Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly” Modified-site 23 /note= “Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Leu, Ser, or Arg” Modified-site 24 /note= “Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu” Modified-site 25 /note= “Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala” Modified-site 26 /note= “Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp” Modified-site 27 /note= “Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala” Modified-site 28 /note= “Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val, or Trp” Modified-site 29 /note= “Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val” Modified-site 30 /note= “Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys” Modified-site 31 /note= “Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln” Modified-site 32 /note= “Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu” Modified-site 33 /note= “Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu” Modified-site 34 /note= “Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile, or Met” Modified-site 35 /note= “Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val” Modified-site 36 /note= “Xaa at position 36 is Asp, Leu, or Val” Modified-site 37 /note= “Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile” Modified-site 38 /note= “Xaa at position 38 is Asn, or Ala” Modified-site 40 /note= “Xaa at position 40 is Leu, Trp, or Arg” Modified-site 41 /note= “Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro” Modified-site 42 /note= “Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met, or Ala” Modified-site 43 /note= “Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly, or Ser” Modified-site 44 /note= “Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala, or Pro” Modified-site 45 /note= “Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu, or His” Modified-site 46 /note= “Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val, or Gly” Modified-site 47 /note= “Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His” Modified-site 48 /note= “Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val, or Asn” Modified-site 49 /note= “Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp” Modified-site 50 /note= “Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met, or Gln” Modified-site 51 /note= “Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His” Modified-site 52 /note= “Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr” Modified-site 53 /note= “Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met” Modified-site 54 /note= “Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala, or Leu” Modified-site 55 /note= “Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly” Modified-site 56 /note= “Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val, or Lys” Modified-site 57 /note= “Xaa at position 57 is Asn or Gly” Modified-site 58 /note= “Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys” Modified-site 59 /note= “Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg” Modified-site 60 /note= “Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr” Modified-site 61 /note= “Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser” Modified-site 62 /note= “Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile” Modified-site 63 /note= “Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val” Modified-site 64 /note= “Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys” Modified-site 65 /note= “Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser” Modified-site 66 /note= “Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser” Modified-site 67 /note= “Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His” Modified-site 68 /note= “Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His” Modified-site 69 /note= “Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu” Modified-site 70 /note= “Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala” Modified-site 71 /note= “Xaa at position 71 is Ala,Met,Leu,Pro,Arg,Glu,Thr,Gln,Trp,or Asn” Modified-site 72 /note= “Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp” Modified-site 73 /note= “Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg” Modified-site 74 /note= “Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, or Ala” Modified-site 75 /note= “Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu” Modified-site 76 /note= “Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp” Modified-site 77 /note= “Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu” Modified-site 78 /note= “Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg” Modified-site 79 /note= “Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp” Modified-site 80 /note= “Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg” Modified-site 81 /note= “Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys” Modified-site 82 /note= “Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met, or Val” Modified-site 83 /note= “Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met” Modified-site 84 /note= “Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val” Modified-site 85 /note= “Xaa at position 85 is Leu, Asn, Val, or Gln” Modified-site 86 /note= “Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys” Modified-site 87 /note= “Xaa at position 87 is Leu, Ser, Trp, or Gly” Modified-site 88 /note= “Xaa at position 88 is Ala, Lys, Arg, Val, or Trp” Modified-site 89 /note= “Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser” Modified-site 90 /note= “Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met” Modified-site 91 /note= “Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His” Modified-site 92 /note= “Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile, or Leu” Modified-site 93 /note= “Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg” Modified-site 94 /note= “Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro” Modified-site 95 /note= “Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr” Modified-site 96 /note= “Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr” Modified-site 97 /note= “Xaa at position 97 is Ile, Val, Lys, Ala, or Asn” Modified-site 98 /note= “Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr, or Pro” Modified-site 99 /note= “Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His” Modified-site 100 /note= “Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro” Modified-site 101 /note= “Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln” Modified-site 102 /note= “Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro” Modified-site 103 /note= “Xaa at position 103 is Asp, or Ser” Modified-site 104 /note= “Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly” Modified-site 105 /note= “Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His” Modified-site 106 /note= “Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro” Modified-site 108 /note= “Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro” Modified-site 109 /note= “Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly” Modified-site 110 /note= “Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, Ala, or Trp” Modified-site 111 /note= “Xaa at position 111 is Leu, Ile, Arg, Asp, or Met” Modified-site 112 /note= “Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe” Modified-site 113 /note= “Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val, or Asn” Modified-site 114 /note= “Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu” Modified-site 115 /note= “Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met” Modified-site 116 /note= “Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile” Modified-site 117 /note= “Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro” Modified-site 118 /note= “Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr” Modified-site 119 /note= “Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg” Modified-site 120 /note= “Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln” Modified-site 121 /note= “Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly” Modified-site 122 /note= “Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys” Modified-site 123 /note= “Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu” 15 Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 133 amino acids amino acid linear peptide Modified-site /note= “Met- may or may not precede the amino acid in position 1” Modified-site 17 /note= “Xaa at position 17 is Ser, Gly, Asp, Met, or Gln” Modified-site 18 /note= “Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln” Modified-site 19 /note= “Xaa at position 19 is Met, Phe, Ile, Arg, or Ala” Modified-site 20 /note= “Xaa at position 20 is Ile or Pro” Modified-site 21 /note; “Xaa at position 21 is Asp or Glu” Modified-site 23 /note= “Xaa at position 23 is Ile, Val, Ala, Leu, or Gly” Modified-site 24 /note= “Xaa at position 24 is Ile, Val, Phe, or Leu” Modified-site 25 /note= “Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala” Modified-site 26 /note= “Xaa at position 26 is His, Phe, Gly, Arg, or Ala” Modified-site 28 /note= “Xaa at position 28 is Lys, Leu, Gln, Gly, Pro, or Val” Modified-site 29 /note= “Xaa at position 29 is Gln, Asn, Leu, Arg, or Val” Modified-site 30 /note= “Xaa at position 30 is Pro, His, Thr, Gly, or Gln” Modified-site 31 /note= “Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln” Modified-site 32 /note= “Xaa at position 32 Leu, Arg, Gln, Asn, Gly, Ala, or Glu” Modified-site 33 /note= “Xaa at position 33 is Pro, Leu, Gln, Ala, or Glu” Modified-site 34 /note= “Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met” Modified-site 35 /note= “Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val” Modified-site 36 /note= “Xaa at position 36 is Asp or Leu” Modified-site 37 /note= “Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile” Modified-site 38 /note= “Xaa at position 38 is Asn or Ala” Modified-site 41 /note= “Xaa at position 41 is Asn, Cys, Arg, His, Met, or Pro” Modified-site 42 /note= “Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, Val, or Arg” Modified-site 44 /note=“Xaa at position 44 is Asp or Glu” Modified-site 45 /note= “Xaa at position 45 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn, Glu, Ser, or Trp” Modified-site 46 /note= “Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val, or Gly” Modified-site 47 /note= “Xaa at position 47 is Ile, Val, or His” Modified-site 49 /note= “Xaa at position 49 is Met, Asn, or Asp” Modified-site 50 /note= “Xaa at position 50 is Glu, Thr, Ala, Asn, Ser, or Asp” Modified-site 51 /note= “Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His” Modified-site 52 /note= “Xaa at position 52 is Asn or Gly” Modified-site 53 /note= “Xaa at position 53 is Leu, Met, or Phe” Modified-site 54 /note= “Xaa at position 54 is Arg Ala, or Ser” Modified-site 55 /note= “Xaa at position 55 is Arg, Thr, Val, Leu, or Gly” Modified-site 56 /note= “Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val, or Lys” Modified-site 59 /note= “Xaa at position 59 is Glu, Tyr, His, Leu, or Arg” Modified-site 60 /note= “Xaa at position 60 is Ala, Ser, Asn, or Thr” Modified-site 61 /note= “Xaa at position 61 is Phe or Ser” Modified-site 62 /note= “Xaa at position 62 is Asn, Val, Pro, Thr, or Ile” Modified-site 63 /note= “Xaa at position 63 is Arg, Tyr, Lys, Ser, His, or Val” Modified-site 64 /note= “Xaa at position 64 is Ala or Asn” Modified-site 65 /note= “Xaa at position 65 is Val, Thr, Leu, or Ser” Modified-site 66 /note= “Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser” Modified-site 67 /note= “Xaa at position 67 is Ser, Phe, Val, Gly, Asn, Ile, or His” Modified-site 68 /note= “Xaa at position 68 is Leu, Val, Ile, Phe, or His” Modified-site 69 /note= “Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly” Modified-site 70 /note= “Xaa at position 70 is Asn or Pro” Modified-site 71 /note= “Xaa at position 71 is Ala, Met, Pro, Arg, Glu, Thr, or Gln” Modified-site 72 /note= “Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp” Modified-site 73 /note= “Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro” Modified-site 74 /note= “Xaa at position 74 is Ile or Met” Modified-site 75 /note= “Xaa at position 75 is Glu, Gly, Asp, Ser, or Gln” Modified-site 76 /note= “Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp” Modified-site 77 /note= “Xaa at position 77 is Ile, Ser, or Leu” Modified-site 79 /note= “Xaa at position 79 is Lys, Thr, Gly, Asn, Met, Arg, Ile, Gly, or Asp” Modified-site 80 /note= “Xaa at position 80 is Asn, Val, Gly, Thr, Leu, Glu, or Arg” Modified-site 81 /note= “Xaa at position 81 is Leu or Val” Modified-site 82 /note= “Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr, or Val” Modified-site 83 /note= “Xaa at position 83 is Pro, Ala, Thr, Trp, or Met” Modified-site 85 /note= “Xaa at position 85 is Leu or Val” Modified-site 87 /note= “Xaa at position 87 is Leu or Ser” Modified-site 88 /note= “Xaa at position 88 is Ala, Arg, or Trp” Modified-site 89 /note= “Xaa at position 89 is Thr, Asp, Glu, His, Asn, or Ser” Modified-site 90 /note= “Xaa at position 90 is Ala, Asp, or Met” Modified-site 91 /note= “Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp” Modified-site 92 /note= “Xaa at position 92 is Pro or Ser” Modified-site 93 /note= “Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg” Modified-site 95 /note= “Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser, or Thr” Modified-site 96 /note= “Xaa at position 96 is Pro or Tyr” Modified-site 97 /note= “Xaa at position 97 is Ile, Val, or Ala” Modified-site 98 /note= “Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Leu, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val, or Pro” Modified-site 99 /note= “Xaa at position 99 is Ile, Leu, Val, or Phe” Modified-site 100 /note= “Xaa at position 100 is Lys, Leu, His, Arg, Ile, Gln, Pro, or Ser” Modified-site 101 /note= “Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu, or Tyr” Modified-site 102 /note= “Xaa at position 102 is Gly, Glu, Lys, or Ser” Modified-site 104 /note= “Xaa at position 104 is Trp, Val, Tyr, Met, or Leu” Modified-site 105 /note= “Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp or His” Modified-site 106 /note= “Xaa at position 106 is Glu, Ser, Ala, or Gly” Modified-site 108 /note= “Xaa at position 108 is Arg, Ala, Gln, Ser, or Lys” Modified-site 109 /note= “Xaa at position 109 is Arg, Thr, Glu, Leu, Ser, or Gly” Modified-site 112 /note= “Xaa at position 112 is Thr, Val, Gln, Glu, His, or Ser” Modified-site 114 /note= “Xaa at position 114 is Tyr or Trp” Modified-site 115 /note= “Xaa at position 115 is Leu or Ala” Modified-site 116 /note= “Xaa at position 116 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr, or Ile” Modified-site 117 /note= “Xaa at position 117 is Thr, Ser, or Asn” Modified-site 119 /note= “Xaa at position 119 is Glu, Ser, Pro, Leu, Thr, or Tyr” Modified-site 120 /note= “Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln” Modified-site 121 /note= “Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly” Modified-site 122 /note= “Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys” Modified-site 123 /note= “Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu” 16 Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Phe Xaa Xaa Lys Leu Xaa 100 105 110 Phe Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 133 amino acids amino acid linear peptide Modified-site /note= “Met- may or may not precede the amino acid in position 1” Modified-site 17 /note= “Xaa at position 17 is Ser, Gly, Asp, Met, or Gln” Modified-site 18 /note= “Xaa at position 18 is Asn, His, or Ile” Modified-site 19 /note= “Xaa at position 19 is Met or Ile” Modified-site 21 /note= “Xaa at position 21 is Asp or Glu” Modified-site 23 /note= “Xaa at position 23 is Ile, Ala, Leu, or Gly” Modified-site 24 /note= “Xaa at position 24 is Ile, Val, or Leu” Modified-site 25 /note= “Xaa at position 25 is Thr, His, Gln, or Ala” Modified-site 26 /note= “Xaa at position 26 is His or Ala” Modified-site 29 /note= “Xaa at position 29 is Gln, Asn, or Val” Modified-site 30 /note= “Xaa at position 30 is Pro, Gly, or Gln” Modified-site 31 /note= “Xaa at position 31 is Pro, Asp, Gly, or Gln” Modified-site 32 /note= “Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu” Modified-site 33 /note= “Xaa at position 33 is Pro or Glu” Modified-site 34 /note= “Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe Thr, or Met” Modified-site 35 /note= “Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val” Modified-site 37 /note= “Xaa at position 37 is Phe, Ser, Pro, or Trp” Modified-site 38 /note= “Xaa at position 38 is Asn or Ala” Modified-site 42 /note= “Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” Modified-site 45 /note= “Xaa at position 45 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser, or Lys” Modified-site 46 /note= “Xaa at position 46 is Asp, Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val, or Cys” Modified-site 50 /note= “Xaa at position 50 is Glu, Ala, Asn, Ser, or Asp” Modified-site 51 /note= “Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His” Modified-site 54 /note= “Xaa at position 54 is Arg or Ala” Modified-site 55 /note= “Xaa at position 55 is Arg, Thr, Val, Leu, or Gly” Modified-site 56 /note= “Xaa at position 56 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val, or Lys” Modified-site 60 /note= “Xaa at position 60 is Ala or Ser” Modified-site 62 /note= “Xaa at position 62 is Asn, Pro, Thr, or Ile” Modified-site 63 /note= “Xaa at position 63 is Arg or Lys” Modified-site 64 /note= “Xaa at position 64 is Ala or Asn” Modified-site 65 /note= “Xaa at position 65 is Val or Thr” Modified-site 66 /note= “Xaa at position 66 is Lys or Arg” Modified-site 67 /note= “Xaa at position 67 is Ser Phe or His” Modified-site 68 /note= “Xaa at position 68 is Leu, Ile, Phe, or His” Modified-site 69 /note= “Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly” Modified-site 71 /note= “Xaa at position 71 is Ala, Pro, or Arg” Modified-site 72 /note= “Xaa at position 72 is Ser, Glu, Arg, or Asp” Modified-site 73 /note= “Xaa at position 73 is Ala or Leu” Modified-site 76 /note= “Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, or Gly” Modified-site 77 /note= “Xaa at position 77 is Ile or Leu” Modified-site 79 /note= “Xaa at position 79 is Lys, Thr, Gly, Asn, Met, Arg, Ile, Gly, or Asp” Modified-site 80 /note= “Xaa at position 80 is Asn, Gly, Glu, or Arg” Modified-site 82 /note= “Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr, or Val” Modified-site 83 /note= “Xaa at position 83 is Pro or Thr” Modified-site 85 /note= “Xaa at position 85 is Leu or Val” Modified-site 87 /note= “Xaa at position 87 is Leu or Ser” Modified-site 88 /note= “Xaa at position 88 is Ala or Trp” Modified-site 91 /note= “Xaa at position 91 is Ala or Pro” Modified-site 93 /note= “Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg” Modified-site 95 /note= “Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser, or Thr” Modified-site 96 /note= “Xaa at position 96 is Pro or Tyr” Modified-site 97 /note= “Xaa at position 97 is Ile or Val” Modified-site 98 /note= “Xaa at position 98 is His, Ile, Asn, Leu, Ala, Thr, Leu, Arg, Gln, Leu, Lys, Met, Ser, Tyr, Val, or Pro” Modified-site 99 /note= “Xaa at position 99 is Ile, Leu, or Val” Modified-site 100 /note= “Xaa at position 100 is Lys, Arg, Ile, Gln, Pro, or Ser” Modified-site 101 /note= “Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Pro, Asn, Ile, Leu, or Tyr” Modified-site 104 /note= “Xaa at position 104 is Trp or Leu” Modified-site 105 /note= “Xaa at position 105 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His” Modified-site 106 /note= “Xaa at position 106 is Glu or Gly” Modified-site 109 /note= “Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser” Modified-site 112 /note= “Xaa at position 112 is Thr, Val, or Gln” Modified-site 114 /note= “Xaa at position 114 is Tyr or Trp” Modified-site 115 /note= “Xaa at position 115 is Leu or Ala” Modified-site 116 /note= “Xaa at position 116 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr, or Ile” Modified-site 117 /note= “Xaa at position 117 is Thr or Ser” Modified-site 120 /note= “Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln” Modified-site 121 /note= “Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly” Modified-site 122 /note= “Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys” Modified-site 123 /note= “Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu” 17 Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15 Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa Xaa Ile Leu 35 40 45 Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa Phe Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu Xaa Xaa Leu Xaa Xaa 65 70 75 80 Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa Xaa Phe Xaa Xaa Lys Leu Xaa 100 105 110 Phe Xaa Xaa Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 133 amino acids amino acid linear peptide Modified-site /note= “Met- may or may not precede the amino acid in position 1” Modified-site 17 /note= “Xaa at position 17 is Ser, Gly, Asp, or Gln” Modified-site 18 /note= “Xaa at position 18 is Asn, His, or Ile” Modified-site 23 /note= “Xaa at position 23 is Ile, Ala, Leu, or Gly” Modified-site 25 /note= “Xaa at position 25 is Thr, His, or Gln” Modified-site 26 /note= “Xaa at position 26 is His or Ala” Modified-site 29 /note=“Xaa at position 29 is Gln or Asn” Modified-site 30 /note= “Xaa at position 30 is Pro or Gly” Modified-site 32 /note= “Xaa at position 32 is Leu, Arg, Asn, or Ala” Modified-site 34 /note= “Xaa at position 34 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met” Modified-site 35 /note= “Xaa at position 35 is Leu, Ala, Asn, or Pro” Modified-site 38 /note= “Xaa at position 38 is Asn or Ala” Modified-site 42 /note= “Xaa at position 42 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” Modified-site 45 /note= “Xaa at position 45 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys” Modified-site 46 /note= “Xaa at position 46 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val, or Thr” Modified-site 50 /note= “Xaa at position 50 is Glu, Asn, Ser, or Asp” Modified-site 51 /note= “Xaa at position 51 is Asn, Arg, Pro, Thr, or His” Modified-site 55 /note= “Xaa at position 55 is Arg, Leu, or Gly” Modified-site 56 /note= “Xaa at position 56 is Pro, Gly, Ser, Ala, Asn, Val, Leu, or Gln” Modified-site 62 /note= “Xaa at position 62 is Asn, Pro, or Thr” Modified-site 64 /note= “Xaa at position 64 is Ala or Asn” Modified-site 65 /note= “Xaa at position 65 is Val or Thr” Modified-site 67 /note= “Xaa at position 67 is Ser or Phe” Modified-site 68 /note= “Xaa at position 68 is Leu or Phe” Modified-site 69 /note= “Xaa at position 69 is Gln, Ala, Glu, or Arg” Modified-site 76 /note= “Xaa at position 76 is Ser, Val, Asn, Pro, or Gly” Modified-site 77 /note= “Xaa at position 77 is Ile or Leu” Modified-site 79 /note= “Xaa at position 79 is Lys, Gly, Asn, Met, Arg, Ile, or Gly” Modified-site 80 /note= “Xaa at position 80 is Asn, Gly, Glu, or Arg” Modified-site 82 /note= “Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr, or Val” Modified-site 87 /note= “Xaa at position 87 is Leu or Ser” Modified-site 88 /note= “Xaa at position 88 is Ala or Trp” Modified-site 91 /note= “Xaa at position 91 is Ala or Pro” Modified-site 93 /note= “Xaa at position 93 is Thr, Asp, or Ala” Modified-site 95 /note= “Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Ser, or Thr” Modified-site 98 /note= “Xaa at position 98 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val, or Leu” Modified-site 99 /note= “Xaa at position 99 is Ile or Leu” Modified-site 100 /note= “Xaa at position 100 is Lys or Arg” Modified-site 101 /note= “Xaa at position 101 is Asp, Pro, Met, Lys, Thr, His, Pro, Asn, Ile, Leu, or Tyr” Modified-site 105 /note= “Xaa at position 105 is Asn, Pro, Ser, Ile, or Asp” Modified-site 108 /note= “Xaa at position 108 is Arg, Ala, or Ser” Modified-site 109 /note= “Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser” Modified-site 112 /note= “Xaa at position 112 is Thr or Gln” Modified-site 116 /note= “Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, Tyr, or Ile” Modified-site 117 /note= “Xaa at position 117 is Thr or Ser” Modified-site 120 /note= “Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln” Modified-site 121 /note= “Xaa at position 121 is Ala, Ser, Ile, Pro, or Asp” Modified-site 122 /note= “Xaa at position 122 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr” Modified-site 123 /note= “Xaa at position 123 is Ala, Met, Glu, Ser, or Leu” 18 Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15 Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa Pro Xaa 20 25 30 Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa Ile Leu 35 40 45 Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala Phe Xaa Arg Xaa 50 55 60 Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu Xaa Xaa Leu Xaa Xaa 65 70 75 80 Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg Xaa Pro 85 90 95 Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa Glu Phe Xaa Xaa Lys Leu Xaa 100 105 110 Phe Tyr Leu Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 111 amino acids amino acid linear peptide Modified-site /note= “Met- or Met-Ala- may or may not precede the amino acid in position 1” Modified-site /note= “Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg” Modified-site /note= “Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln” Modified-site /note= “Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys” Modified-site /note= “Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala” Modified-site /note= “Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser, or Val” Modified-site /note= “Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly” Modified-site /note= “Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Leu, Ser or Arg” Modified-site 10 /note= “Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu” Modified-site 11 /note= “Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala” Modified-site 12 /note= “Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp” Modified-site 13 /note= “Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala” Modified-site 14 /note= “Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val, or Trp” Modified-site 15 /note= “Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val” Modified-site 16 /note= “Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys” Modified-site 17 /note= “Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln” Modified-site 18 /note= “Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu” Modified-site 19 /note= “Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu” Modified-site 20 /note= “Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile, or Met” Modified-site 21 /note= “Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val” Modified-site 22 /note= “Xaa at position 22 is Asp, Leu, or Val” Modified-site 23 /note= “Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile” Modified-site 24 /note= “Xaa at position 24 is Asn or Ala” Modified-site 26 /note= “Xaa at position 26 is Leu, Trp, or Arg” Modified-site 27 /note= “Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, or Pro” Modified-site 28 /note= “Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile, or Met” Modified-site 29 /note= “Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly, or Ser” Modified-site 30 /note= “Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala, or Pro” Modified-site 31 /note= “Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His, or Trp” Modified-site 32 /note= “Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val, or Gly” Modified-site 33 /note= “Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His” Modified-site 34 /note= “Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val, or Asn” Modified-site 35 /note= “Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp” Modified-site 36 /note= “Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met, or Gln” Modified-site 37 /note= “Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His” Modified-site 38 /note= “Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr” Modified-site 39 /note= “Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met” Modified-site 40 /note= “Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala, or Leu” Modified-site 41 /note= “Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly” Modified-site 42 /note= “Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val, or Lys” Modified-site 43 /note= “Xaa at position 43 is Asn or Gly” Modified-site 44 /note= “Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys” Modified-site 45 /note= “Xaa at position 45 is Glu, Tyr, His, Leu, Pro, or Arg” Modified-site 46 /note= “Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr” Modified-site 47 /note= “Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser” Modified-site 48 /note= “Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile” Modified-site 49 /note= “Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val” Modified-site 50 /note= “Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys” Modified-site 51 /note= “Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser” Modified-site 52 /note= “Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser” Modified-site 53 /note= “Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His” Modified-site 54 /note= “Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His” Modified-site 55 /note= “Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu” Modified-site 56 /note= “Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala” Modified-site 57 /note= “Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn” Modified-site 58 /note= “Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp” Modified-site 59 /note= “Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg” Modified-site 60 /note= “Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala” Modified-site 61 /note= “Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu” Modified-site 62 /note= “Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp” Modified-site 63 /note= “Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu” Modified-site 64 /note= “Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg” Modified-site 65 /note= “Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp” Modified-site 66 /note= “Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg” Modified-site 67 /note= “Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys” Modified-site 68 /note= “Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met, or Val” Modified-site 69 /note= “Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met” Modified-site 70 /note= “Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val” Modified-site 71 /note= “Xaa at position 71 is Leu, Asn, Val, or Gln” Modified-site 72 /note= “Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys” Modified-site 73 /note= “Xaa at position 73 is Leu, Ser, Trp, or Gly” Modified-site 74 /note= “Xaa at position 74 is Ala, Lys, Arg, Val, or Trp” Modified-site 75 /note= “Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser” Modified-site 76 /note= “Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met” Modified-site 77 /note= “Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His” Modified-site 78 /note= “Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile, or Leu” Modified-site 79 /note= “Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg” Modified-site 80 /note= “Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro” Modified-site 81 /note= “Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr” Modified-site 82 /note= “Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr” Modified-site 83 /note= “Xaa at position 83 is Ile, Val, Lys, Ala, or Asn” Modified-site 84 /note= “Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr, or Pro” Modified-site 85 /note= “Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His” Modified-site 86 /note= “Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro” Modified-site 87 /note= “Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln” Modified-site 88 /note= “Xaa at position 88 Gly, Leu, Glu, Lys, Ser, Tyr, or Pro” Modified-site 89 /note= “Xaa at position 89 is Asp or Ser” Modified-site 90 /note= “Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly” Modified-site 91 /note= “Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His” Modified-site 92 /note= “Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro” Modified-site 94 /note= “Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro” Modified-site 95 /note= “Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly” Modified-site 96 /note= “Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala, or Trp” Modified-site 97 /note= “Xaa at position 97 is Leu, Ile, Arg, Asp, or Met” Modified-site 98 /note= “Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe” Modified-site 99 /note= “Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val, or Asn” Modified-site 100 /note= “Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu” Modified-site 101 /note= “Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met” Modified-site 102 /note= “Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile” Modified-site 103 /note= “Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro” Modified-site 104 /note= “Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr” Modified-site 105 /note= “Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg” Modified-site 106 /note= “Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val or Gln” Modified-site 107 /note= “Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly” Modified-site 108 /note= “Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys” Modified-site 109 /note= “Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu” 19 Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Gln 100 105 110 111 amino acids amino acid linear peptide Modified-site /note= “Met- or Met-Ala- may or may not precede the amino acid in position 1” Modified-site /note= “Xaa at position 3 is Ser, Gly, Asp, Met, or Gln” Modified-site /note= “Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln” Modified-site /note= “Xaa at position 5 is Met, Phe, Ile, Arg, or Ala” Modified-site /note= “Xaa at position 6 is Ile or Pro” Modified-site /note= “Xaa at position 7 is Asp or Glu” Modified-site /note= “Xaa at position 9 is Ile, Val, Ala, Leu, or Gly” Modified-site 10 /note= “Xaa at position 10 is Ile, Val, Phe, or Leu” Modified-site 11 /note= “Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala” Modified-site 12 /note= “Xaa at position 12 is His, Phe, Gly, Arg, or Ala” Modified-site 14 /note= “Xaa at position 14 is Lys, Leu, Gln, Gly, Pro, or Val” Modified-site 15 /note= “Xaa at position 15 is Gln, Asn, Leu, Arg, or Val” Modified-site 16 /note= “Xaa at position 16 is Pro, His, Thr, Gly, or Gln” Modified-site 17 /note= “Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln” Modified-site 18 /note= “Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala or Glu” Modified-site 19 /note= “Xaa at poisiton 19 is Pro, Leu, Gln, Ala, or Glu” Modified-site 20 /note= “Xaa at positon 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met” Modified-site 21 /note= “Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val” Modified-site 22 /note= “Xaa at position 22 is Asp or Leu” Modified-site 23 /note= “Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile” Modified-site 24 /note= “Xaa at position 24 is Asn or Ala” Modified-site 27 /note= “Xaa at position 27 is Asn, Cys, Arg, His, Met, or Pro” Modified-site 28 /note= “Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” Modified-site 30 /note= “Xaa at position 30 is Asp or Glu” Modified-site 31 /note= “Xaa at position 31 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn, Glu, Ser, or Trp” Modified-site 32 /note= “Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val, or Gly” Modified-site 33 /note= “Xaa at position 33 is Ile, Val, or His” Modified-site 35 /note= “Xaa at position 35 is Met, Asn, or Asp” Modified-site 36 /note= “Xaa at position 36 is Glu, Thr, Ala, Asn, Ser, or Asp” Modified-site 37 /note= “Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His” Modified-site 38 /note= “Xaa at position 38 is Asn or Gly” Modified-site 39 /note= “Xaa at position 39 is Leu, Met, or Phe” Modified-site 40 /note= “Xaa at position 40 is Arg, Ala, or Ser” Modified-site 41 /note= “Xaa at position 41 is Arg, Thr, Val, Leu, or Gly” Modified-site 42 /note= “Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val, or Lys” Modified-site 45 /note= “Xaa at position 45 is Glu, Tyr, His, Leu, or Arg” Modified-site 46 /note= “Xaa at position 46 is Ala, Ser, Asn, or Thr” Modified-site 47 /note= “Xaa at position 47 is Phe or Ser” Modified-site 48 /note= “Xaa at position 48 is Asn, Val, Pro, Thr, or Ile” Modified-site 49 /note= “Xaa at position 49 is Arg, Tyr, Lys, Ser, His, or Val” Modified-site 50 /note= “Xaa at position 50 is Ala or Asn” Modified-site 51 /note= “Xaa at position 51 is Val, Thr, Leu, or Ser” Modified-site 52 /note= “Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser” Modified-site 53 /note= “Xaa at position 53 is Ser, Phe, Val, Gly, Asn, Ile, or His” Modified-site 54 /note= “Xaa at position 54 is Leu, Val, Ile, Phe, or His” Modified-site 55 /note= “Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly” Modified-site 56 /note= “Xaa at position 56 is Asn or Pro” Modified-site 57 /note= “Xaa at position 57 is Ala, Met, Pro, Arg, Glu, Thr, or Gln” Modified-site 58 /note= “Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp” Modified-site 59 /note= “Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro” Modified-site 60 /note= “Xaa at position 60 is Ile or Met” Modified-site 61 /note= “Xaa at position 61 is Glu, Gly, Asp, Ser, or Gln” Modified-site 62 /note= “Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp” Modified-site 63 /note= “Xaa at position 63 is Ile, Ser, or Leu” Modified-site 65 /note= “Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp” Modified-site 66 /note= “Xaa at position 66 is Asn, Val, Gly, Thr, Leu, Glu, or Arg” Modified-site 67 /note= “Xaa at position 67 is Leu or Val” Modified-site 68 /note= “Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr, or Val” Modified-site 69 /note= “Xaa at position 69 is Pro, Ala, Thr, Trp, or Met” Modified-site 71 /note= “Xaa at position 71 is Leu or Val” Modified-site 73 /note= “Xaa at position 73 is Leu or Ser” Modified-site 74 /note= “Xaa at position 74 is Ala, Arg, or Trp” Modified-site 75 /note= “Xaa at position 75 is Thr, Asp, Glu, His, Asn, or Ser” Modified-site 76 /note= “Xaa at position 76 is Ala, Asp, or Met” Modified-site 77 /note= “Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp” Modified-site 78 /note= “Xaa at position 78 is Pro or Ser” Modified-site 79 /note= “Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg” Modified-site 81 /note= “Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser, or Thr” Modified-site 82 /note= “Xaa at position 82 is Pro or Tyr” Modified-site 83 /note= “Xaa at position 83 is Ile, Val, or Ala” Modified-site 84 /note= “Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val, or Pro” Modified-site 85 /note= “Xaa at position 85 is Ile, Leu, Val, or Phe” Modified-site 86 /note= “Xaa at position 86 is Lys, Leu, His, Arg, Ile, Gln, Pro, or Ser” Modified-site 87 /note= “Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu, or Tyr” Modified-site 88 /note= “Xaa at position 88 is Gly, Glu, Lys, or Ser” Modified-site 90 /note= “Xaa at position 90 is Trp, Val, Tyr, Met, or Leu” Modified-site 91 /note= “Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His” Modified-site 92 /note= “Xaa at position 92 is Glu, Ser, Ala, or Gly” Modified-site 94 /note= “Xaa at position 94 is Arg, Ala, Gln, Ser, or Lys” Modified-site 95 /note= “Xaa at position 95 Arg, Thr, Glu, Leu, Ser, or Gly” Modified-site 98 /note= “Xaa at position 98 is Thr, Val, Gln, Glu, His, or Ser” Modified-site 100 /note= “Xaa at position 100 is Tyr or Trp” Modified-site 101 /note= “Xaa at position 101 is Leu or Ala” Modified-site 102 /note= “Xaa at position 102 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr, or Ile” Modified-site 103 /note= “Xaa at position 103 is Thr, Ser, or Asn” Modified-site 105 /note= “Xaa at position 105 is Glu, Ser, Pro, Leu, Thr, or Tyr” Modified-site 106 /note= “Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln” Modified-site 107 /note= “Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly” Modified-site 108 /note= “Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys” Modified-site 109 /note= “Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu” 20 Asn Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa Xaa 20 25 30 Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu 50 55 60 Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Phe Xaa Xaa Lys 85 90 95 Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Gln Gln 100 105 110 111 amino acids amino acid linear peptide Modified-site /note= “Met- or Met-Ala- may or may not precede the amino acid in position 1” Modified-site /note= “Xaa at position 3 is Ser, Gly, Asp, Met, or Gln” Modified-site /note= “Xaa at position 4 is Asn, His, or Ile” Modified-site /note= “Xaa at position 5 is Met or Ile” Modified-site /note= “Xaa at position 7 is Asp or Glu” Modified-site /note= “Xaa at position 9 is Ile, Ala, Leu, or Gly” Modified-site 10 /note= “Xaa at position 10 is Ile, Val, or Leu” Modified-site 11 /note= “Xaa at position 11 is Thr, His, Gln, or Ala” Modified-site 12 /note= “Xaa at position 12 is His or Ala” Modified-site 15 /note= “Xaa at position 15 is Gln, Asn, or Val” Modified-site 16 /note= “Xaa at position 16 is Pro, Gly, or Gln” Modified-site 17 /note= “Xaa at position 17 is Pro, Asp, Gly, or Gln” Modified-site 18 /note= “Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu” Modified-site 19 /note= “Xaa at position 19 is Pro or Glu” Modified-site 20 /note= “Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met” Modified-site 21 /note= “Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val” Modified-site 23 /note= “Xaa at position 23 is Phe, Ser, Pro, or Trp” Modified-site 24 /note= “Xaa at position 24 is Asn or Ala” Modified-site 28 /note= “Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” Modified-site 30 /note= “Xaa at position 30 is Asp or Glu” Modified-site 31 /note= “Xaa at position 31 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser, or Lys” Modified-site 32 /note= “Xaa at position 32 is Asp, Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val, or Cys” Modified-site 36 /note= “Xaa at position 36 is Glu, Ala, Asn, Ser, or Asp” Modified-site 37 /note= “Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His” Modified-site 40 /note= “Xaa at position 40 is Arg or Ala” Modified-site 41 /note= “Xaa at position 41 is Arg, Thr, Val, Leu, or Gly” Modified-site 42 /note= “Xaa at position 42 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val, or Lys” Modified-site 46 /note= “Xaa at position 46 is Ala or Ser” Modified-site 48 /note= “Xaa at position 48 is Asn, Pro, Thr, or Ile” Modified-site 49 /note= “Xaa at position 49 is Arg or Lys” Modified-site 50 /note= “Xaa at position 50 is Ala or Asn” Modified-site 51 /note= “Xaa at position 51 is Val or Thr” Modified-site 52 /note= “Xaa at position 52 is Lys or Arg” Modified-site 53 /note= “Xaa at position 53 is Ser, Phe, or His” Modified-site 54 /note= “Xaa at position 54 is Leu, Ile, Phe, or His” Modified-site 55 /note= “Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly” Modified-site 57 /note= “Xaa at position 57 is Ala, Pro, or Arg” Modified-site 58 /note= “Xaa at position 58 is Ser, Glu, Arg, or Asp” Modified-site 59 /note= “Xaa at position 59 is Ala or Leu” Modified-site 62 /note= “Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, or Gly” Modified-site 63 /note= “Xaa at position 63 is Ile or Leu” Modified-site 65 /note= “Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, Gly, or Asp” Modified-site 66 /note= “Xaa at position 66 is Asn, Gly, Glu, or Arg” Modified-site 68 /note= “Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr, or Val” Modified-site 69 /note= “Xaa at position 69 is Pro or Thr” Modified-site 71 /note= “Xaa at position 71 is Leu or Val” Modified-site 73 /note= “Xaa at position 73 is Leu or Ser” Modified-site 74 /note= “Xaa at position 74 is Ala or Trp” Modified-site 77 /note= “Xaa at position 77 is Ala or Pro” Modified-site 79 /note= “Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg” Modified-site 81 /note= “Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser, or Thr” Modified-site 82 /note= “Xaa at position 82 is Pro or Tyr” Modified-site 83 /note= “Xaa at position 83 is Ile or Val” Modified-site 84 /note= “Xaa at position 84 is His, Ile, Asn, Leu, Ala, Thr, Leu, Arg, Gln, Leu, Lys, Met, Ser, Tyr, Val, or Pro” Modified-site 85 /note= “Xaa at position 85 is Ile, Leu, or Val” Modified-site 86 /note= “Xaa at position 86 is Lys, Arg, Ile, Gln, Pro, or Ser” Modified-site 87 /note= “Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Asn, Ile, Leu, or Tyr” Modified-site 90 /note= “Xaa at position 90 is Trp or Leu” Modified-site 91 /note=“Xaa at position 91 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His” Modified-site 92 /note= “Xaa at position 92 is Glu or Gly” Modified-site 94 /note= “Xaa at position 94 is Arg, Ala, or Ser” Modified-site 95 /note= “Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser” Modified-site 98 /note= “Xaa at position 98 is Thr, Val, or Gln” Modified-site 100 /note= “Xaa at position 100 is Tyr or Trp” Modified-site 101 /note= “Xaa at position 101 is Leu or Ala” Modified-site 102 /note= “Xaa at position 102 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr, or Ile” Modified-site 103 /note= “Xaa at position 103 is Thr or Ser” Modified-site 106 /note= “Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln” Modified-site 107 /note= “Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly” Modified-site 108 /note= “Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys” Modified-site 109 /note= “Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu” 21 Asn Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa Xaa 20 25 30 Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa Phe Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu Xaa Xaa Leu 50 55 60 Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa Xaa Phe Xaa Xaa Lys 85 90 95 Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln 100 105 110 111 amino acids amino acid linear peptide Modified-site /note= “Met- or Met-Ala- may or may not precede the amino acid in position 1” Modified-site /note= “Xaa at position 3 is Ser, Gly, Asp, or Gln” Modified-site /note= “Xaa at position 4 is Asn, His, or Ile” Modified-site /note= “Xaa at position 9 is Ile, Ala, Leu, or Gly” Modified-site 11 /note= “Xaa at position 11 is Thr, His, or Gln” Modified-site 12 /note= “Xaa at position 12 is His or Ala” Modified-site 15 /note= “Xaa at position 15 is Gln or Asn” Modified-site 16 /note= “Xaa at position 16 is Pro or Gly” Modified-site 18 /note= “Xaa at position 18 is Leu, Arg, Asn, or Ala” Modified-site 20 /note= “Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met” Modified-site 21 /note= “Xaa at position 21 is Leu, Ala, Asn, or Pro” Modified-site 24 /note= “Xaa at position 24 is Asn or Ala” Modified-site 28 /note= “Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” Modified-site 31 /note= “Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys” Modified-site 32 /note= “Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val, or Thr” Modified-site 36 /note= “Xaa at position 36 is Glu, Asn, Ser, or Asp” Modified-site 37 /note= “Xaa at position 37 is Asn, Arg, Pro, Thr, or His” Modified-site 41 /note= “Xaa at position 41 is Arg, Leu, or Gly” Modified-site 42 /note= “Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu, or Gln” Modified-site 48 /note= “Xaa at position 48 is Asn, Pro, or Thr” Modified-site 50 /note= “Xaa at position 50 is Ala or Asn” Modified-site 51 /note= “Xaa at position 51 is Val or Thr” Modified-site 53 /note= “Xaa at position 53 is Ser or Phe” Modified-site 54 /note= “Xaa at position 54 is Leu or Phe” Modified-site 55 /note= “Xaa at position 55 is Gln, Ala, Glu, or Arg” Modified-site 62 /note= “Xaa at position 62 is Ser, Val, Asn, Pro, or Gly” Modified-site 63 /note= “Xaa at position 63 is Ile or Leu” Modified-site 65 /note= “Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly” Modified-site 66 /note= “Xaa at position 66 is Asn, Gly, Glu, or Arg” Modified-site 68 /note= “Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr, or Val” Modified-site 73 /note= “Xaa at position 73 is Leu or Ser” Modified-site 74 /note= “Xaa at position 74 is Ala or Trp” Modified-site 77 /note= “Xaa at position 77 is Ala or Pro” Modified-site 79 /note= “Xaa at position 79 is Thr, Asp, or Ala” Modified-site 81 /note= “Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser, or Thr” Modified-site 84 /note= “Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val, or Leu” Modified-site 85 /note= “Xaa at position 85 is Ile or Leu” Modified-site 86 /note= “Xaa at position 86 is Lys or Arg” Modified-site 87 /note= “Xaa at position 87 is Asp, Pro, Met, Lys, His, Pro, Asn, Ile, Leu, or Tyr” Modified-site 91 /note= “Xaa at position 91 is Asn, Pro, Ser, Ile, or Asp” Modified-site 94 /note= “Xaa at position 94 is Arg, Ala, or Ser” Modified-site 95 /note= “Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser” Modified-site 98 /note= “Xaa at position 98 is Thr or Gln” Modified-site 102 /note= “Xaa at position 102 is Lys, Val, Trp, or Ile” Modified-site 103 /note= “Xaa at position 103 is Thr, Ala, His, Phe, Tyr, or Ser” Modified-site 106 /note= “Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln” Modified-site 107 /note= “Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp” Modified-site 108 /note= “Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr” Modified-site 109 /note= “Xaa at position 109 is Ala, Met, Glu, Ser, or Leu” 22 Asn Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa 1 5 10 15 Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa 20 25 30 Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala Phe Xaa 35 40 45 Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu Xaa Xaa Leu 50 55 60 Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg 65 70 75 80 Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa Glu Phe Xaa Xaa Lys 85 90 95 Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln 100 105 110 111 amino acids amino acid linear peptide Modified-site /note= “Met- or Met-Ala- may or may not precede the amino acid in position 1” 23 Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro 1 5 10 15 Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp 20 25 30 Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn 35 40 45 Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu 50 55 60 Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg 65 70 75 80 His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys 85 90 95 Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 100 105 110 58 base pairs nucleic acid single linear DNA (synthetic) 24 CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG 58 58 base pairs nucleic acid single linear DNA (synthetic) 25 TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG 58 20 base pairs nucleic acid single linear DNA (synthetic) 26 TGTCTGCTCA GGCCATGGCT 20 67 base pairs nucleic acid single linear DNA (synthetic) 27 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGTCGAC TTATTACGTG GGTGCGGCCG 60 TGGCTAG 67 55 base pairs nucleic acid single linear DNA (synthetic) 28 GCGCGAATTC ATTCCAGTCA CCGTCGACTT ATTAGATTGG ATGTCGCGTG GGTGC 55 16 base pairs nucleic acid single linear DNA (synthetic) 29 TGAACCATAT GTCAGG 16 24 base pairs nucleic acid single linear DNA (synthetic) 30 AATTCCTGAC ATATGGTTCA TGCA 24 43 base pairs nucleic acid single linear DNA (synthetic) 31 GCGCGAATTC GTCGACTTAT TAGTCCTTGA TATGGATTGG ATG 43 67 base pairs nucleic acid single linear DNA (synthetic) 32 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG ATGSNNCGTG GGTGCGGCCG 60 TGGCTAG 67 64 base pairs nucleic acid single linear DNA (synthetic) 33 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG SNNTCGCGTG GGTGCGGCCG 60 TGGC 64 61 base pairs nucleic acid single linear DNA (synthetic) 34 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATSNN ATGTCGCGTG GGTGCGGCCG 60 T 61 58 base pairs nucleic acid single linear DNA (synthetic) 35 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGSNNTGG ATGTCGCGTG GGTGCGGC 58 24 base pairs nucleic acid single linear DNA (synthetic) 36 GATATGGATT GGATGTCGCG TGGG 24 55 base pairs nucleic acid single linear DNA (synthetic) 37 GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TSNNGATTGG ATGTCGCGTG GGTGC 55 52 base pairs nucleic acid single linear DNA (synthetic) 38 GCGCGAATTC ATTCCAGTCA CCGTCCTTSN NATGGATTGG ATGTCGCGTG GG 52 49 base pairs nucleic acid single linear DNA (synthetic) 39 GCGCGAATTC ATTCCAGTCA CCGTCSNNGA TATGGATTGG ATGTCGCGT 49 46 base pairs nucleic acid single linear DNA (synthetic) 40 GCGCGAATTC ATTCCAGTCA CCSNNCTTGA TATGGATTGG ATGTCG 46 24 base pairs nucleic acid single linear DNA (synthetic) 41 GTCACCGTCC TTGATATGGA TTGG 24 43 base pairs nucleic acid single linear DNA (synthetic) 42 GCGCGAATTC ATTCCAGTCS NNGTCCTTGA TATGGATTGG ATG 43 40 base pairs nucleic acid single linear DNA (synthetic) 43 GCGCGAATTC ATTCCASNNA CCGTCCTTGA TATGGATTGG 40 37 base pairs nucleic acid single linear DNA (synthetic) 44 GCGCGAATTC ATTSNNGTCA CCGTCCTTGA TATGGAT 37 34 base pairs nucleic acid single linear DNA (synthetic) 45 GCGCGAATTC SNNCCAGTCA CCGTCCTTGA TATG 34 25 base pairs nucleic acid single linear DNA (synthetic) 46 GAATTCATTC CAGTCACCGT TCCTT 25 26 base pairs nucleic acid single linear DNA (synthetic) 47 CGCGCGGAAT TCATTCCAGT CACCGT 26 42 base pairs nucleic acid single linear DNA (synthetic) 48 CGCGCGCCAT GGCTAACTGC ATTATAACAC ACACTTAAAG CA 42 58 base pairs nucleic acid single linear DNA (synthetic) 49 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAACA GCCACCTTTG CCTTTGCT 58 76 base pairs nucleic acid single linear DNA (synthetic) 50 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCTGG 60 ACTTCAACAA CCTCAA 76 50 base pairs nucleic acid single linear DNA (synthetic) 51 GCGCGCGATA TCTTGGTCTT CTTCACCATT CAGCGGCAGC GGTGGCTGCT 50 65 base pairs nucleic acid single linear DNA (synthetic) 52 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATGAGGT TGTTGAAGTC 60 CAGCA 65 47 base pairs nucleic acid single linear DNA (synthetic) 53 GCGCGCCTCG AGGTTTGGAC GACGAAGATC TTGGTCTTCA CCATTGA 47 68 base pairs nucleic acid single linear DNA (synthetic) 54 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGT TATTTTCCAT 60 CAGGATAT 68 50 base pairs nucleic acid single linear DNA (synthetic) 55 GCGCGCTGAT GCATTCTGCA GAGACTTGAC GAGGTTTGGA CGACGAAGGT 50 47 base pairs nucleic acid single linear DNA (synthetic) 56 GCGCGCCTCG AGGCATTCAA CCGTGCTGCA TCAGCAATTG AGAGCAT 47 35 base pairs nucleic acid single linear DNA (synthetic) 57 GCGCGCCTGC AGAATATTCT TAAAAATCTC CTGCC 35 53 base pairs nucleic acid single linear DNA (synthetic) 58 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCCCATGTC TGCCGCTAGC CAC 53 71 base pairs nucleic acid single linear DNA (synthetic) 59 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GACGGCCGCA 60 CCCACGCGAC A 71 67 base pairs nucleic acid single linear DNA (synthetic) 60 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCA GGGCAGACAT 60 GGCAGGA 67 78 base pairs nucleic acid single linear DNA (synthetic) 61 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTAT 60 TCCAGTCACC GTCCTTGA 78 60 base pairs nucleic acid single linear DNA (synthetic) 62 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA CAGTTTACGA CGGAATTCAT 60 42 base pairs nucleic acid single linear DNA (synthetic) 63 CGCGCGAAGC TTATTACTGT TGGGTTTTCA GATAGAAGGT CA 42 58 base pairs nucleic acid single linear DNA (synthetic) 64 CGCGCGCCAT GGCTAACTGC NNSAACATGA TCGATGAAAT TATAACACAC TTAAAGCA 58 111 amino acids amino acid linear peptide 65 Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro 1 5 10 15 Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp 20 25 30 Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn 35 40 45 Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu 50 55 60 Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg 65 70 75 80 His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys 85 90 95 Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 100 105 110 113 amino acids amino acid linear peptide 66 Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys 1 5 10 15 Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp 20 25 30 Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala 35 40 45 Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser 50 55 60 Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro 65 70 75 80 Thr Arg His Pro Ile Ile Ile Arg Asp Gly Asp Trp Asn Glu Phe Arg 85 90 95 Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln 100 105 110 Gln 113 amino acids amino acid linear peptide 67 Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys 1 5 10 15 Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp 20 25 30 Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala 35 40 45 Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser 50 55 60 Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro 65 70 75 80 Thr Arg Arg Pro Ile Ile Ile Arg Asp Gly Asp Trp Asn Glu Phe Arg 85 90 95 Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln 100 105 110 Gln 339 base pairs nucleic acid double linear DNA (genomic) 68 ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60 CCGCTGCTGG ACTTCAACAA CCTCAATGAC GAAGACATGT CTATCCTGAT GGAAAATAAC 120 CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA AGTCTCTGCA GAATGCATCA 180 GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240 ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300 TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG 339 113 amino acids amino acid linear peptide 69 Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys 1 5 10 15 Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Asp Glu Asp 20 25 30 Met Ser Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala 35 40 45 Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser 50 55 60 Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro 65 70 75 80 Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg 85 90 95 Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln 100 105 110 Gln 45 base pairs nucleic acid single linear DNA (synthetic) 70 CTTTAAGTGT GTTATAATTT CGTTGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 71 CATGGCTAAC TGCTCTAACA TGATCCAAGA AATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 72 CTTTAAGTGT GTTATAATTT CTTGGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 73 CATGGCTAAC TGCTCTAACA TGATCGAAGA AATTATAACA 40 76 base pairs nucleic acid single linear DNA (synthetic) 74 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACANNS TTAAAGCAGC 60 CACCTTTGCC TTTGCT 76 76 base pairs nucleic acid single linear DNA (synthetic) 75 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC NNSAAGCAGC 60 CACCTTTGCC TTTGCT 76 76 base pairs nucleic acid single linear DNA (synthetic) 76 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTANNSCAGC 60 CACCTTTGCC TTTGCT 76 94 base pairs nucleic acid single linear DNA (synthetic) 77 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGNNSC 60 CACCTTTGCC TTTGCTGGAC TTCAACAACC TCAA 94 94 base pairs nucleic acid single linear DNA (synthetic) 78 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGN 60 NSCCTTTGCC TTTGCTGGAC TTCAACAACC TCAA 94 94 base pairs nucleic acid single linear DNA (synthetic) 79 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60 CANNSTTGCC TTTGCTGGAC TTCAACAACC TCAA 94 94 base pairs nucleic acid single linear DNA (synthetic) 80 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60 CACCTNNSCC TTTGCTGGAC TTCAACAACC TCAA 94 94 base pairs nucleic acid single linear DNA (synthetic) 81 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60 CACCTTTGNN STTGCTGGAC TTCAACAACC TCAA 94 94 base pairs nucleic acid single linear DNA (synthetic) 82 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60 CACCTTTGCC TNNSCTGGAC TTCAACAACC TCAA 94 65 base pairs nucleic acid single linear DNA (synthetic) 83 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAG TCSNNCAGCG GCAGCGGTGG 60 CTGCT 65 65 base pairs nucleic acid single linear DNA (synthetic) 84 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAS NNCAGCAGCG GCAGCGGTGG 60 CTGCT 65 65 base pairs nucleic acid single linear DNA (synthetic) 85 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTSNNG TCCAGCAGCG GCAGCGGTGG 60 CTGCT 65 65 base pairs nucleic acid single linear DNA (synthetic) 86 GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTSNNGAAG TCCAGCAGCG GCAGCGGTGG 60 CTGCT 65 65 base pairs nucleic acid single linear DNA (synthetic) 87 GCGCGCGATA TCTTGGTCTT CACCATTGAG SNNGTTGAAG TCCAGCAGCG GCAGCGGTGG 60 CTGCT 65 65 base pairs nucleic acid single linear DNA (synthetic) 88 GCGCGCGATA TCTTGGTCTT CACCATTSNN GTTGTTGAAG TCCAGCAGCG GCAGCGGTGG 60 CTGCT 65 83 base pairs nucleic acid single linear DNA (synthetic) 89 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCACC 60 SNNGAGGTTG TTGAAGTCCA GCA 83 83 base pairs nucleic acid single linear DNA (synthetic) 90 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCSNN 60 ATTGAGGTTG TTGAAGTCCA GCA 83 83 base pairs nucleic acid single linear DNA (synthetic) 91 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCSNNACC 60 ATTGAGGTTG TTGAAGTCCA GCA 83 83 base pairs nucleic acid single linear DNA (synthetic) 92 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GSNNTTCACC 60 ATTGAGGTTG TTGAAGTCCA GCA 83 83 base pairs nucleic acid single linear DNA (synthetic) 93 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCSN NGTCTTCACC 60 ATTGAGGTTG TTGAAGTCCA GCA 83 83 base pairs nucleic acid single linear DNA (synthetic) 94 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATSNNTT GGTCTTCACC 60 ATTGAGGTTG TTGAAGTCCA GCA 83 65 base pairs nucleic acid single linear DNA (synthetic) 95 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGSNNATCTT GGTCTTCACC 60 ATTGA 65 65 base pairs nucleic acid single linear DNA (synthetic) 96 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATS NNGATATCTT GGTCTTCACC 60 ATTGA 65 65 base pairs nucleic acid single linear DNA (synthetic) 97 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCSNNC AGGATATCTT GGTCTTCACC 60 ATTGA 65 65 base pairs nucleic acid single linear DNA (synthetic) 98 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTSNNCATC AGGATATCTT GGTCTTCACC 60 ATTGA 65 65 base pairs nucleic acid single linear DNA (synthetic) 99 GCGCGCCTCG AGGTTTGGAC GACGAAGGTT SNNTTCCATC AGGATATCTT GGTCTTCACC 60 ATTGA 65 65 base pairs nucleic acid single linear DNA (synthetic) 100 GCGCGCCTCG AGGTTTGGAC GACGAAGSNN ATTTTCCATC AGGATATCTT GGTCTTCACC 60 ATTGA 65 86 base pairs nucleic acid single linear DNA (synthetic) 101 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG 60 ACGSNNGTTA TTTTCCATCA GGATAT 86 86 base pairs nucleic acid single linear DNA (synthetic) 102 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG 60 SNNAAGGTTA TTTTCCATCA GGATAT 86 86 base pairs nucleic acid single linear DNA (synthetic) 103 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGSNN 60 ACGAAGGTTA TTTTCCATCA GGATAT 86 86 base pairs nucleic acid single linear DNA (synthetic) 104 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTSNNACG 60 ACGAAGGTTA TTTTCCATCA GGATAT 86 86 base pairs nucleic acid single linear DNA (synthetic) 105 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GSNNTGGACG 60 ACGAAGGTTA TTTTCCATCA GGATAT 86 86 base pairs nucleic acid single linear DNA (synthetic) 106 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCSN NGTTTGGACG 60 ACGAAGGTTA TTTTCCATCA GGATAT 86 68 base pairs nucleic acid single linear DNA (synthetic) 107 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCSNNGA GGTTTGGACG 60 ACGAAGGT 68 68 base pairs nucleic acid single linear DNA (synthetic) 108 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AASNNCTCGA GGTTTGGACG 60 ACGAAGGT 68 68 base pairs nucleic acid single linear DNA (synthetic) 109 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTS NNTGCCTCGA GGTTTGGACG 60 ACGAAGGT 68 68 base pairs nucleic acid single linear DNA (synthetic) 110 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGSNNG AATGCCTCGA GGTTTGGACG 60 ACGAAGGT 68 68 base pairs nucleic acid single linear DNA (synthetic) 111 GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCSNNGTTG AATGCCTCGA GGTTTGGACG 60 ACGAAGGT 68 68 base pairs nucleic acid single linear DNA (synthetic) 112 GCGCGCTGAT GCATTCTGCA GAGACTTGAC SNNACGGTTG AATGCCTCGA GGTTTGGACG 60 ACGAAGGT 68 65 base pairs nucleic acid single linear DNA (synthetic) 113 GCGCGCCTCG AGGCATTCAA CCGTGCTNNS AAGTCTCTGC AGAATGCATC AGCAATTGAG 60 AGCAT 65 65 base pairs nucleic acid single linear DNA (synthetic) 114 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC NNSTCTCTGC AGAATGCATC AGCAATTGAG 60 AGCAT 65 65 base pairs nucleic acid single linear DNA (synthetic) 115 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGNNSCTGC AGAATGCATC AGCAATTGAG 60 AGCAT 65 65 base pairs nucleic acid single linear DNA (synthetic) 116 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTNNSC AGAATGCATC AGCAATTGAG 60 AGCAT 65 65 base pairs nucleic acid single linear DNA (synthetic) 117 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGN NSAATGCATC AGCAATTGAG 60 AGCAT 65 65 base pairs nucleic acid single linear DNA (synthetic) 118 GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGC AGNNSGCATC AGCAATTGAG 60 AGCAT 65 53 base pairs nucleic acid single linear DNA (synthetic) 119 GCGCGCCTGC AGAATNNSTC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCC 53 53 base pairs nucleic acid single linear DNA (synthetic) 120 GCGCGCCTGC AGAATGCANN SGCAATTGAG AGCATTCTTA AAAATCTCCT GCC 53 53 base pairs nucleic acid single linear DNA (synthetic) 121 GCGCGCCTGC AGAATGCATC ANNSATTGAG AGCATTCTTA AAAATCTCCT GCC 53 53 base pairs nucleic acid single linear DNA (synthetic) 122 GCGCGCCTGC AGAATGCATC AGCANNSGAG AGCATTCTTA AAAATCTCCT GCC 53 53 base pairs nucleic acid single linear DNA (synthetic) 123 GCGCGCCTGC AGAATGCATC AGCAATTNNS AGCATTCTTA AAAATCTCCT GCC 53 53 base pairs nucleic acid single linear DNA (synthetic) 124 GCGCGCCTGC AGAATGCATC AGCAATTGAG NNSATTCTTA AAAATCTCCT GCC 53 71 base pairs nucleic acid single linear DNA (synthetic) 125 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCNNSCTTA AAAATCTCCT GCCATGTCTG 60 CCGCTAGCCA C 71 71 base pairs nucleic acid single linear DNA (synthetic) 126 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTNNSA AAAATCTCCT GCCATGTCTG 60 CCGCTAGCCA C 71 71 base pairs nucleic acid single linear DNA (synthetic) 127 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTN NSAATCTCCT GCCATGTCTG 60 CCGCTAGCCA C 71 134 amino acids amino acid linear peptide 128 Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn 1 5 10 15 Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro 20 25 30 Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile 35 40 45 Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg 50 55 60 Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys 65 70 75 80 Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His 85 90 95 Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu 100 105 110 Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr 115 120 125 Leu Ser Leu Ala Ile Phe 130 133 amino acids amino acid linear peptide Modified-site /note= “Met- may or may not precede the amino acid in position 1” Modified-site 18 /note= “Xaa at position 18 is Asn or Ile” Modified-site 25 /note= “Xaa at position 25 is Thr or His” Modified-site 29 /note= “Xaa at position 29 is Gln, Arg, or Val” Modified-site 32 /note= “Xaa at position 32 is Leu, Ala, or Asn” Modified-site 37 /note= “Xaa at position 37 is Phe, Pro, or Ser” Modified-site 42 /note= “Xaa at position 42 is Glu, Ala, or Ser” Modified-site 45 /note= “Xaa at position 45 is Gln, Val, or Met” Modified-site 51 /note= “Xaa at position 51 is Asn or Arg” Modified-site 55 /note= “Xaa at position 55 is Arg, Leu, or Thr” Modified-site 59 /note= “Xaa at position 59 is Glu or Leu” Modified-site 60 /note= “Xaa at position 60 is Ala or Ser” Modified-site 62 /note= “Xaa at position 62 is Asn or Val” Modified-site 67 /note= “Xaa at position 67 is Ser, Asn, or His” Modified-site 69 /note= “Xaa at position 69 is Gln or Glu” Modified-site 73 /note= “Xaa at position 73 is Ala or Gly” Modified-site 76 /note= “Xaa at position 76 is Ser or Ala” Modified-site 79 /note= “Xaa at position 79 is Lys or Arg” Modified-site 82 /note= “Xaa at position 82 is Leu, Glu, or Val” Modified-site 87 /note= “Xaa at position 87 is Leu or Ser” Modified-site 93 /note= “Xaa at position 93 is Pro or Ser” Modified-site 98 /note= “Xaa at position 98 is His, Ile, or Thr” Modified-site 101 /note= “Xaa at position 101 is Asp or Ala” Modified-site 105 /note= “Xaa at position 105 is Asn or Glu” Modified-site 109 /note= “Xaa at position 109 is Arg or Glu” Modified-site 116 /note= “Xaa at position 116 is Lys or Val” Modified-site 120 /note= “Xaa at position 120 is Asn, Gln, or His” Modified-site 123 /note= “Xaa at position 123 is Ala or Glu” 129 Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15 Ser Xaa Met Ile Asp Glu Ile Ile Xaa His Leu Lys Xaa Pro Pro Xaa 20 25 30 Pro Leu Leu Asp Xaa Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu 35 40 45 Met Glu Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg Ala 50 55 60 Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu Xaa Asn 65 70 75 80 Leu Xaa Pro Cys Leu Pro Xaa Ala Thr Ala Ala Pro Xaa Arg His Pro 85 90 95 Ile Xaa Ile Lys Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr 100 105 110 Phe Tyr Leu Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 111 amino acids amino acid linear peptide Modified-site /note= “Met- or Met-Ala may or may not precede the amino acid in position 1” Modified-site /note= “Xaa at position 4 is Asn or Ile” Modified-site 11 /note= “Xaa at position 11 is Thr or His” Modified-site 15 /note= “Xaa at position 15 is Gln, Arg, or Val” Modified-site 18 /note= “Xaa at position 18 is Leu, Ala, or Asn” Modified-site 23 /note= “Xaa at position 23 is Phe, Pro, or Ser” Modified-site 28 /note= “Xaa at position 28 is Glu, Ala, or Ser” Modified-site 31 /note= “Xaa at position 31 is Gln, Val, or Met” Modified-site 37 /note= “Xaa at position 37 is Asn or Arg” Modified-site 41 /note= “Xaa at position 41 is Arg, Leu, or Thr” Modified-site 45 /note= “Xaa at position 45 is Glu or Leu” Modified-site 46 /note= “Xaa at position 46 is Ala or Ser” Modified-site 48 /note= “Xaa at position 48 is Asn or Val” Modified-site 53 /note= “Xaa at position 53 is Ser, Asn, or His” Modified-site 55 /note= “Xaa at position 55 is Gln or Glu” Modified-site 59 /note= “Xaa at position 59 is Ala or Gly” Modified-site 62 /note= “Xaa at position 62 is Ser or Ala” Modified-site 65 /note= “Xaa at position 65 is Lys or Arg” Modified-site 68 /note= “Xaa at position 68 is Leu, Glu, or Val” Modified-site 73 /note= “Xaa at position 73 is Leu or Ser” Modified-site 79 /note= “Xaa at position 79 is Pro or Ser” Modified-site 84 /note= “Xaa at position 84 is His, Ile, or Thr” Modified-site 87 /note= “Xaa at position 87 is Asp or Ala” Modified-site 91 /note= “Xaa at position 91 is Asn or Glu” Modified-site 95 /note= “Xaa at position 95 is Arg or Glu” Modified-site 102 /note= “Xaa at position 102 is Lys or Val” Modified-site 106 /note= “Xaa at position 106 is Asn, Gln, or His” Modified-site 109 /note= “Xaa at position 109 is Ala or Glu” 130 Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile Xaa His Leu Lys Xaa Pro 1 5 10 15 Pro Xaa Pro Leu Leu Asp Xaa Asn Asn Leu Asn Xaa Glu Asp Xaa Asp 20 25 30 Ile Leu Met Glu Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa 35 40 45 Arg Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu 50 55 60 Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr Ala Ala Pro Xaa Arg 65 70 75 80 His Pro Ile Xaa Ile Lys Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys 85 90 95 Leu Thr Phe Tyr Leu Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln 100 105 110 58 base pairs nucleic acid single linear DNA (synthetic) 131 CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG 58 58 base pairs nucleic acid single linear DNA (synthetic) 132 TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG 58 29 base pairs nucleic acid double linear DNA (genomic) 133 AAGGAGATAT ATCCATGAAC TGCTCTAAC 29 5 amino acids amino acid linear peptide 134 Met Asn Cys Ser Asn 1 5 120 amino acids amino acid linear peptide 135 Met Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 1 5 10 15 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 20 25 30 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 35 40 45 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 50 55 60 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 65 70 75 80 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 85 90 95 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 100 105 110 Thr Thr Leu Arg Leu Ala Ile Phe 115 120 71 base pairs nucleic acid single linear DNA (synthetic) 136 AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGA 60 CCACTCTGTC G 71 71 base pairs nucleic acid single linear DNA (synthetic) 137 CTAGCGACAG AGTGGTCTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60 GTTTACGACG G 71 71 base pairs nucleic acid single linear DNA (synthetic) 138 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AANNSCTCCT GCCATGTCTG 60 CCGCTAGCCA C 71 71 base pairs nucleic acid single linear DNA (synthetic) 139 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATNNSCT GCCATGTCTG 60 CCGCTAGCCA C 71 71 base pairs nucleic acid single linear DNA (synthetic) 140 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCNN SCCATGTCTG 60 CCGCTAGCCA C 71 89 base pairs nucleic acid single linear DNA (synthetic) 141 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GNNSTGTCTG 60 CCGCTAGCCA CGGCCGCACC CACGCGACA 89 89 base pairs nucleic acid single linear DNA (synthetic) 142 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCANNSCTG 60 CCGCTAGCCA CGGCCGCACC CACGCGACA 89 89 base pairs nucleic acid single linear DNA (synthetic) 143 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTNNS 60 CCGCTAGCCA CGGCCGCACC CACGCGACA 89 408 base pairs nucleic acid double linear DNA (genomic) 144 ATGGCTCCAA TGACTCAGAC TACTTCTCTT AAGACTTCTT GGGTTAACTG CTCTAACATG 60 ATCGATGAAA TTATAACACA CTTAAAGCAG CCACCTTTGC CTTTGCTGGA CTTCAACAAC 120 CTCAATGGGG AAGACCAAGA CATTCTGATG GAAAATAACC TTCGAAGGCC AAACCTGGAG 180 GCATTCAACA GGGCTGTCAA GAGTTTACAG AATGCATCAG CAATTGAGAG CATTCTTAAA 240 AATCTCCTGC CATGTCTGCC CCTGGCCACG GCCGCACCCA CGCGACATCC AATCCATATC 300 AAGGACGGTG ACTGGAATGA ATTCCGTCGT AAACTGACCT TCTATCTGAA AACCTTGGAG 360 AACGCGCAGG CTCAACAGAC CACTCTGTCG CTAGCGATCT TTTAATAA 408 157 base pairs nucleic acid double linear DNA (genomic) CDS 1..156 145 ATC GAT GAA ATC ATC ACC CAC CTG AAG CAG CCA CCG CTG CCG CTG CTG 48 Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu 1 5 10 15 GAC TTC AAC AAC CTC AAT GGT GAA GAC CAA GAT ATC CTG ATG GAA AAT 96 Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn 20 25 30 AAC CTT CGT CGT CCA AAC CTC GAG GCA TTC AAC CGT GCT GTC AAG TCT 144 Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser 35 40 45 CTG CAG AAT GCA T 157 Leu Gln Asn Ala 50 52 amino acids amino acid linear protein 146 Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu 1 5 10 15 Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn 20 25 30 Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser 35 40 45 Leu Gln Asn Ala 50 414 base pairs nucleic acid double linear DNA (genomic) 147 CCATGGCTCC AATGACTCAG ACTACTTCTC TTAAGACTTC TTGGGTTAAC TGCTCTAACA 60 TGATCGATGA AATTATAACA CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTCAACA 120 ACCTCAATGG GGAAGACCAA GACATTCTGA TGGAAAATAA CCTTCGAAGG CCAAACCTGG 180 AGGCATTCAA CAGGGCTGTC AAGAGTTTAC AGAATGCATC AGCAATTGAG AGCATTCTTA 240 AAAATCTCCT GCCATGTCTG CCCCTGGCCA CGGCCGCACC CACGCGACAT CCAATCCATA 300 TCAAGGACGG TGACTGGAAT GAATTCCGTC GTAAACTGAC CTTCTATCTG AAAACCTTGG 360 AGAACGCGCA GGCTCAACAG ACCACTCTGT CGCTAGCGAT CTTTTAATAA GCTT 414 414 base pairs nucleic acid double linear DNA (genomic) 148 AAGCTTATTA AAAGATCGCT AGCGACAGAG TGGTCTGTTG AGCCTGCGCG TTCTCCAAGG 60 TTTTCAGATA GAAGGTCAGT TTACGACGGA ATTCATTCCA GTCACCGTCC TTGATATGGA 120 TTGGATGTCG CGTGGGTGCG GCCGTGGCCA GGGGCAGACA TGGCAGGAGA TTTTTAAGAA 180 TGCTCTCAAT TGCTGATGCA TTCTGTAAAC TCTTGACAGC CCTGTTGAAT GCCTCCAGGT 240 TTGGCCTTCG AAGGTTATTT TCCATCAGAA TGTCTTGGTC TTCCCCATTG AGGTTGTTGA 300 AGTCCAGCAA AGGCAAAGGT GGCTGCTTTA AGTGTGTTAT AATTTCATCG ATCATGTTAG 360 AGCAGTTAAC CCAAGAAGTC TTAAGAGAAG TAGTCTGAGT CATTGGAGCC ATGG 414 81 base pairs nucleic acid double linear DNA (genomic) 149 ATGATGATTA CTCTGCGCAA ACTTCCTCTG GCGGTTGCCG TCGCAGCGGG CGTAATGTCT 60 GCTCAGGCCA TGGCTAACTG C 81 81 base pairs nucleic acid double linear DNA (genomic) 150 GCAGTTAGCC ATGGCCTGAG CAGACATTAC GCCCGCTGCG ACGGCAACCG CCAGAGGAAG 60 TTTGCGCAGA GTAATCATCA T 81 24 base pairs nucleic acid single linear DNA (synthetic) 151 CATGGCTAAC TGCTCTAACA TGAT 24 22 base pairs nucleic acid single linear DNA (synthetic) 152 CGATCATGTT AGAGCAGTTA GC 22 12 base pairs nucleic acid double linear DNA (genomic) 153 ATGGCTAACT GC 12 4 amino acids amino acid linear peptide 154 Met Ala Asn Cys 1 34 base pairs nucleic acid single linear DNA (synthetic) 155 GCCGATACCG CGGCATACTC CCACCATTCA GAGA 34 33 base pairs nucleic acid single linear DNA (synthetic) 156 GCCGATAAGA TCTAAAACGG GTATGGAGAA ACA 33 89 base pairs nucleic acid single linear DNA (synthetic) 157 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60 NNSCTAGCCA CGGCCGCACC CACGCGACA 89 89 base pairs nucleic acid single linear DNA (synthetic) 158 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60 CCGNNSGCCA CGGCCGCACC CACGCGACA 89 89 base pairs nucleic acid single linear DNA (synthetic) 159 GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60 CCGCTANNSA CGGCCGCACC CACGCGACA 89 86 base pairs nucleic acid single linear DNA (synthetic) 160 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCGGC 60 SNNGGCCAGG GGCAGACATG GCAGGA 86 86 base pairs nucleic acid single linear DNA (synthetic) 161 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCSNN 60 CGTGGCCAGG GGCAGACATG GCAGGA 86 86 base pairs nucleic acid single linear DNA (synthetic) 162 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGSNNGGC 60 CGTGGCCAGG GGCAGACATG GCAGGA 86 86 base pairs nucleic acid single linear DNA (synthetic) 163 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TSNNTGCGGC 60 CGTGGCCAGG GGCAGACATG GCAGGA 86 86 base pairs nucleic acid single linear DNA (synthetic) 164 CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGSN NGGGTGCGGC 60 CGTGGCCAGG GGCAGACATG GCAGGA 86 48 base pairs nucleic acid single linear DNA (synthetic) 165 TTTCAGATAG AAGGTCAGTT TACGACGGAA SNNATTCCAG TCACCGTC 48 48 base pairs nucleic acid single linear DNA (synthetic) 166 TTTCAGATAG AAGGTCAGTT TACGACGSNN TTCATTCCAG TCACCGTC 48 48 base pairs nucleic acid single linear DNA (synthetic) 167 TTTCAGATAG AAGGTCAGTT TACGSNNGAA TTCATTCCAG TCACCGTC 48 48 base pairs nucleic acid single linear DNA (synthetic) 168 TTTCAGATAG AAGGTCAGTT TSNNACGGAA TTCATTCCAG TCACCGTC 48 48 base pairs nucleic acid single linear DNA (synthetic) 169 TTTCAGATAG AAGGTCAGSN NACGACGGAA TTCATTCCAG TCACCGTC 48 48 base pairs nucleic acid single linear DNA (synthetic) 170 TTTCAGATAG AAGGTSNNTT TACGACGGAA TTCATTCCAG TCACCGTC 48 23 base pairs nucleic acid single linear DNA (synthetic) 171 CGCGCGAAGC TTATTACTGT TGA 23 78 base pairs nucleic acid single linear DNA (synthetic) 172 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAASNNCA 60 GTTTACGACG GAATTCAT 78 78 base pairs nucleic acid single linear DNA (synthetic) 173 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TASNNGGTCA 60 GTTTACGACG GAATTCAT 78 78 base pairs nucleic acid single linear DNA (synthetic) 174 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGS NNGAAGGTCA 60 GTTTACGACG GAATTCAT 78 78 base pairs nucleic acid single linear DNA (synthetic) 175 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTSNNA TAGAAGGTCA 60 GTTTACGACG GAATTCAT 78 78 base pairs nucleic acid single linear DNA (synthetic) 176 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTSNNCAGA TAGAAGGTCA 60 GTTTACGACG GAATTCAT 78 78 base pairs nucleic acid single linear DNA (synthetic) 177 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA SNNTTTCAGA TAGAAGGTCA 60 GTTTACGACG GAATTCAT 78 60 base pairs nucleic acid single linear DNA (synthetic) 178 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCSNN GGTTTTCAGA TAGAAGGTCA 60 60 base pairs nucleic acid single linear DNA (synthetic) 179 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTSNNCAA GGTTTTCAGA TAGAAGGTCA 60 60 base pairs nucleic acid single linear DNA (synthetic) 180 CGCGCGAAGC TTATTACTGT TGAGCCTGCG CSNNCTCCAA GGTTTTCAGA TAGAAGGTCA 60 60 base pairs nucleic acid single linear DNA (synthetic) 181 CGCGCGAAGC TTATTACTGT TGAGCCTGSN NGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60 60 base pairs nucleic acid single linear DNA (synthetic) 182 CGCGCGAAGC TTATTACTGT TGAGCSNNCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60 60 base pairs nucleic acid single linear DNA (synthetic) 183 CGCGCGAAGC TTATTACTGT TGSNNCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60 24 base pairs nucleic acid single linear DNA (synthetic) 184 TGCTCTAACA TGATCGATGA AATT 24 24 base pairs nucleic acid single linear DNA (synthetic) 185 GAAATTATAA CACACTTAAA GCAG 24 24 base pairs nucleic acid single linear DNA (synthetic) 186 AAGCAGCCAC CTTTGCCTTT GCTG 24 24 base pairs nucleic acid single linear DNA (synthetic) 187 AAGCAGCCAC CGCTGCCGCT GCTG 24 24 base pairs nucleic acid single linear DNA (synthetic) 188 CTCAATGGTG AAGACCAAGA TATC 24 24 base pairs nucleic acid single linear DNA (synthetic) 189 GATATCCTGA TGGAAAATAA CCTT 24 24 base pairs nucleic acid single linear DNA (synthetic) 190 AACCTTCGTC GTCCAAACCT CGAG 24 24 base pairs nucleic acid single linear DNA (synthetic) 191 CTCGAGGCAT TCAACCGTGC TGTC 24 24 base pairs nucleic acid single linear DNA (synthetic) 192 GCTGTCAAGT CTCTGCAGAA TGCA 24 24 base pairs nucleic acid single linear DNA (synthetic) 193 AATGCATCAG CAATTGAGAG CATT 24 24 base pairs nucleic acid single linear DNA (synthetic) 194 AGCATTCTTA AAAATCTCCT GCCA 24 24 base pairs nucleic acid single linear DNA (synthetic) 195 CTGCCATGTC TGCCCCTGGC CACG 24 24 base pairs nucleic acid single linear DNA (synthetic) 196 CTGGCCACGG CCGCACCCAC GCGA 24 24 base pairs nucleic acid single linear DNA (synthetic) 197 AATGAATTCC GTCGTAAACT GACC 24 24 base pairs nucleic acid single linear DNA (synthetic) 198 CTGACCTTCT ATCTGAAAAC CTTG 24 24 base pairs nucleic acid single linear DNA (synthetic) 199 ACCTTGGAGA ACGCGCAGGC TCAA 24 22 base pairs nucleic acid single linear DNA (synthetic) 200 GAATGCATCA GCAATTGAGA GC 22 20 base pairs nucleic acid single linear DNA (synthetic) 201 AATTGCTGAT GCATTCTGCA 20 24 base pairs nucleic acid single linear DNA (synthetic) 202 ATTCTTAAAA ATCTCCTGCC ATGT 24 24 base pairs nucleic acid single linear DNA (synthetic) 203 CAGGAGATTT TTAAGAATGC TCTC 24 30 base pairs nucleic acid single linear DNA (synthetic) 204 CTGCCCCTGG CCACGGCCGC ACCCACGCGA 30 30 base pairs nucleic acid single linear DNA (synthetic) 205 GGGTGCGGCC GTGGCCAGGG GCAGACATGG 30 34 base pairs nucleic acid single linear DNA (synthetic) 206 CATCCAATCA TCATCCGTGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 207 AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGATGTC GCGT 44 34 base pairs nucleic acid single linear DNA (synthetic) 208 CGCCCAATCA TCATCCGTGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 209 AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGGCGTC GCGT 44 40 base pairs nucleic acid single linear DNA (synthetic) 210 CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 211 CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC 45 36 base pairs nucleic acid single linear DNA (synthetic) 212 CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 213 GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 214 AACAACCTCA ATGACGAAGA CATGTCT 27 18 base pairs nucleic acid single linear DNA (synthetic) 215 AGACATGTCT TCGTCATT 18 339 base pairs nucleic acid double linear DNA (genomic) 216 ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60 CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC 120 CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA 180 GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240 ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300 TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG 339 111 amino acids amino acid linear peptide 217 Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro 1 5 10 15 Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp 20 25 30 Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn 35 40 45 Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu 50 55 60 Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg 65 70 75 80 His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys 85 90 95 Leu Thr Phe Tyr Leu Trp Thr Leu Glu Asn Ala Gln Ala Gln Gln 100 105 110 111 amino acids amino acid linear peptide 218 Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro 1 5 10 15 Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp 20 25 30 Ile Leu Met Asp Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn 35 40 45 Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu 50 55 60 Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg 65 70 75 80 His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys 85 90 95 Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 100 105 110 45 base pairs nucleic acid single linear DNA (synthetic) 219 CTTTAAGTGT GTTATAATTT CTTCGATCAT GTTAGAGCAG TTAGC 45 339 base pairs nucleic acid double linear DNA (genomic) 220 ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60 CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC 120 CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA 180 GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240 ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300 TTCTATCTGT GGACCTTGGA GAACGCGCAG GCTCAACAG 339 40 base pairs nucleic acid single linear DNA (synthetic) 221 CATGGCTAAC TGCTCTAACA TGATCAACGA AATTATAACA 40 58 base pairs nucleic acid single linear DNA (synthetic) 222 CGCGCGCCAT GGCTAACTGC TCTNNSATGA TCGATGAAAT TATAACACAC TTAAAGCA 58 58 base pairs nucleic acid single linear DNA (synthetic) 223 CGCGCGCCAT GGCTAACTGC TCTAACNNSA TCGATGAAAT TATAACACAC TTAAAGCA 58 58 base pairs nucleic acid single linear DNA (synthetic) 224 CGCGCGCCAT GGCTAACTGC TCTAACATGN NSGATGAAAT TATAACACAC TTAAAGCA 58 58 base pairs nucleic acid single linear DNA (synthetic) 225 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCNNSGAAAT TATAACACAC TTAAAGCA 58 58 base pairs nucleic acid single linear DNA (synthetic) 226 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATNNSAT TATAACACAC TTAAAGCA 58 76 base pairs nucleic acid single linear DNA (synthetic) 227 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAANN SATAACACAC TTAAAGCAGC 60 CACCTTTGCC TTTGCT 76 76 base pairs nucleic acid single linear DNA (synthetic) 228 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TNNSACACAC TTAAAGCAGC 60 CACCTTTGCC TTTGCT 76 76 base pairs nucleic acid single linear DNA (synthetic) 229 CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATANNSCAC TTAAAGCAGC 60 CACCTTTGCC TTTGCT 76 40 base pairs nucleic acid single linear DNA (synthetic) 230 CATGGCTAAC TGCTCTAACA TGATCAGCGA AATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 231 CTTTAAGTGT GTTATAATTT CGCTGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 232 CATGGCTAAC TGCTCTAACA TGATCACCGA AATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 233 CTTTAAGTGT GTTATAATTT CCGTGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 234 CATGGCTAAC TGCTCTAACA TGATCGATAA CATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 235 CTTTAAGTGT GTTATAATGT TATCGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 236 CATGGCTAAC TGCTCTAACA TGATCGATGA CATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 237 CTTTAAGTGT GTTATAATGT CATCGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 238 CATGGCTAAC TGCTCTAACA TGATCGATCA GATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 239 CTTTAAGTGT GTTATAATCT GATCGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 240 CATGGCTAAC TGCTCTAACA TGATCGATCT GATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 241 CTTTAAGTGT GTTATAATCA GATCGATCAT GTTAGAGCAG TTAGC 45 40 base pairs nucleic acid single linear DNA (synthetic) 242 CATGGCTAAC TGCTCTAACA TGATCGATGT TATTATAACA 40 45 base pairs nucleic acid single linear DNA (synthetic) 243 CTTTAAGTGT GTTATAATAA CATCGATCAT GTTAGAGCAG TTAGC 45 36 base pairs nucleic acid single linear DNA (synthetic) 244 CACTTAAAGC AGCCACCTTT GCCTGCTCTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 245 GAGGTTGTTG AAGTCCAGAG CAGGCAAAGG TGGCTG 36 36 base pairs nucleic acid single linear DNA (synthetic) 246 CACTTAAAGC AGCCACCTTT GCCTCGTCTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 247 GAGGTTGTTG AAGTCCAGAC GAGGCAAAGG TGGCTG 36 36 base pairs nucleic acid single linear DNA (synthetic) 248 CACTTAAAGC AGCCACCTTT GCCTCAGCTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 249 GAGGTTGTTG AAGTCCAGCT GAGGCAAAGG TGGCTG 36 36 base pairs nucleic acid single linear DNA (synthetic) 250 CACTTAAAGC AGCCACCTTT GCCTGAACTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 251 GAGGTTGTTG AAGTCCAGCT CAGGCAAAGG TGGCTG 36 36 base pairs nucleic acid single linear DNA (synthetic) 252 CACTTAAAGC AGCCACCTTT GCCTATCCTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 253 GAGGTTGTTG AAGTCCAGGA TAGGCAAAGG TGGCTG 36 36 base pairs nucleic acid single linear DNA (synthetic) 254 CACTTAAAGC AGCCACCTTT CCCTTTCCTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 255 GAGGTTGTTG AAGTCCAGGA AAGGCAAAGG TGGCTG 36 36 base pairs nucleic acid single linear DNA (synthetic) 256 CACTTAAAGC AGCCACCTTT GCCTACCCTG GACTTC 36 36 base pairs nucleic acid single linear DNA (synthetic) 257 GAGGTTGTTG AAGTCCAGGG TAGGCAAAGG TGGCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 258 AACAACCTCA ATCGTGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 259 ATCTTGGTCT TCACGATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 260 AACAACCTCA ATAACGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 261 ATCTTGGTCT TCGTTATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 262 AACAACCTCA ATGAAGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 263 ATCTTGGTCT TCTTCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 264 AACAACCTCA ATATCGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 265 ATCTTGGTCT TCGATATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 266 AACAACCTCA ATCTGGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 267 ATCTTGGTCT TCCAGATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 268 AACAACCTCA ATAAAGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 269 ATCTTGGTCT TCTTTATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 270 AACAACCTCA ATATGGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 271 ATCTTGGTCT TCCATATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 272 AACAACCTCA ATTTCGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 273 ATCTTGGTCT TCGAAATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 274 AACAACCTCA ATACCGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 275 ATCTTGGTCT TCGGTATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 276 AACAACCTCA ATTACGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 277 ATCTTGGTCT TCGTAATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 278 AACAACCTCA ATGTTGAAGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 279 ATCTTGGTCT TCAACATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 280 AACAACCTCA ATGGGCGTGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 281 ATCTTGGTCT CGCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 282 AACAACCTCA ATGGGCAGGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 283 ATCTTGGTCC TGCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 284 AACAACCTCA ATGGGGGTGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 285 ATCTTGGTCA CCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 286 AACAACCTCA ATGGGACCGA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 287 ATCTTGGTCG GTCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 288 AACAACCTCA ATGGGGAAGC TCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 289 ATCTTGAGCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 290 AACAACCTCA ATGGGGAAAA CCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 291 ATCTTGGTTT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 292 AACAACCTCA ATGGGGAACA GCAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 293 ATCTTGCTGT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 294 AACAACCTCA ATGGGGAAGA ACAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 295 ATCTTGTTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 296 AACAACCTCA ATGGGGAAGA CGCTGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 297 ATCAGCGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 298 AACAACCTCA ATGGGGAAGA CCGTGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 299 ATCACGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 300 AACAACCTCA ATGGGGAAGA CAACGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 301 ATCGTTGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 302 AACAACCTCA ATGGGGAAGA CGACGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 303 ATCGTCGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 304 AACAACCTCA ATGGTGAAGA CGAAGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 305 ATCTTCGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 306 AACAACCTCA ATGGTGAAGA CCACGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 307 ATCGTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 308 AACAACCTCA ATGGGGAAGA CATCGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 309 ATCGATGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 310 AACAACCTCA ATGGGGAAGA CTCCGAT 27 18 base pairs nucleic acid single linear DNA (synthetic) 311 ATCGGAGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 312 AACAACCTCA ATGGGGAAGA CCAAGCT 27 18 base pairs nucleic acid single linear DNA (synthetic) 313 AGCTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 314 AACAACCTCA ATGGGGAAGA CCAAAAC 27 18 base pairs nucleic acid single linear DNA (synthetic) 315 GTTTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 316 AACAACCTCA ATGGGGAAGA CCAACAG 27 18 base pairs nucleic acid single linear DNA (synthetic) 317 CTGTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 318 AACAACCTCA ATGGGGAAGA CCAAGAA 27 18 base pairs nucleic acid single linear DNA (synthetic) 319 TTCTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 320 AACAACCTCA ATGGGGAAGA CCAACAC 27 18 base pairs nucleic acid single linear DNA (synthetic) 321 GTGTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 322 AACAACCTCA ATGGGGAAGA CCAAATC 27 18 base pairs nucleic acid single linear DNA (synthetic) 323 GATTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 324 AACAACCTCA ATGGGGAAGA CCAACTG 27 18 base pairs nucleic acid single linear DNA (synthetic) 325 CAGTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 326 AACAACCTCA ATGGGGAAGA CCAAAAA 27 18 base pairs nucleic acid single linear DNA (synthetic) 327 TTTTTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 328 AACAACCTCA ATGGGGAAGA CCAATAC 27 18 base pairs nucleic acid single linear DNA (synthetic) 329 GTATTGGTCT TCCCCATT 18 27 base pairs nucleic acid single linear DNA (synthetic) 330 AACAACCTCA ATGGGGAAGA CCAAGTT 27 18 base pairs nucleic acid single linear DNA (synthetic) 331 AACTTGGTCT TCCCCATT 18 36 base pairs nucleic acid single linear DNA (synthetic) 332 ATCGCTATGG AAAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 333 CCTTCGAAGG TTATTTTCCA TAGCGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 334 ATCGAAATGG AAAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 335 CCTTCGAAGG TTATTTTCCA TTTCGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 336 ATCAAAATGG AAAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 337 CCTTCGAAGG TTATTTTCCA TTTTGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 338 ATCATGATGG AAAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 339 CCTTCGAAGG TTATTTTCCA TCATGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 340 ATCACCATGG AAAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 341 CCTTCGAAGG TTATTTTCCA TGGTGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 342 ATCGTTATGG AAAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 343 CCTTCGAAGG TTATTTTCCA TAACGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 344 ATCCTGATGC ACAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 345 CCTTCGAAGG TTATTGTGCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 346 ATCCTGATGA TGAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 347 CCTTCGAAGG TTATTCATCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 348 ATCCTGATGT TCAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 349 CCTTCGAAGG TTATTGAACA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 350 ATCCTGATGG CTAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 351 CCTTCGAAGG TTATTAGCCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 352 ATCCTGATGA ACAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 353 CCTTCGAAGG TTATTGTTCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 354 ATCCTGATGA TCAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 355 CCTTCGAAGG TTATTGATCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 356 ATCCTGATGA AAAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 357 CCTTCGAAGG TTATTTTTCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 358 ATCCTGATGT CCAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 359 CCTTCGAAGG TTATTGGACA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 360 ATCCTGATGG TTAATAACCT TCGAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 361 CCTTCGAAGG TTATTAACCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 362 ATCCTGATGG AAAATAACCT TGCTAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 363 CCTAGCAAGG TTATTTTCCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 364 ATCCTGATGG AAAATAACCT TAACAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 365 CCTGTTAAGG TTATTTTCCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 366 ATCCTGATGG AAAATAACCT TCACAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 367 CCTGTGAAGG TTATTTTCCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 368 ATCCTGATGG AAAATAACCT TAAAAGGCCA AACCTG 36 27 base pairs nucleic acid single linear DNA (synthetic) 369 CCTTTTAAGG TTATTTTCCA TCAGGAT 27 36 base pairs nucleic acid single linear DNA (synthetic) 370 ATCCTGATGG AAAATAACCT TCGAAGGGCT AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 371 CCTGTTGAAT GCCTCCAGGT TAGC 24 36 base pairs nucleic acid single linear DNA (synthetic) 372 ATCCTGATGG AAAATAACCT TCGAAGGCGT AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 373 CCTGTTGAAT GCCTCCAGGT TACG 24 36 base pairs nucleic acid single linear DNA (synthetic) 374 ATCCTGATGG AAAATAACCT TCGAAGGAAC AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 375 CCTGTTGAAT GCCTCCAGGT TGTT 24 36 base pairs nucleic acid single linear DNA (synthetic) 376 ATCCTGATGG AAAATAACCT TCGAAGGGAA AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 377 CCTGTTGAAT GCCTCCAGGT TTTC 24 36 base pairs nucleic acid single linear DNA (synthetic) 378 ATCCTGATGG AAAATAACCT TCGAAGGCAC AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 379 CCTGTTGAAT GCCTCCAGGT TGTG 24 36 base pairs nucleic acid single linear DNA (synthetic) 380 ATCCTGATGG AAAATAACCT TCGAAGGCTG AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 381 CCTGTTGAAT GCCTCCAGGT TCAG 24 36 base pairs nucleic acid single linear DNA (synthetic) 382 ATCCTGATGG AAAATAACCT TCGAAGGTTC AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 383 CCTGTTGAAT GCCTCCAGGT TGAA 24 36 base pairs nucleic acid single linear DNA (synthetic) 384 ATCCTGATGG AAAATAACCT TCGAAGGACC AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 385 CCTGTTGAAT GCCTCCAGGT TGGT 24 36 base pairs nucleic acid single linear DNA (synthetic) 386 ATCCTGATGG AAAATAACCT TCGAAGGTAC AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 387 CCTGTTGAAT GCCTCCAGGT TGTA 24 36 base pairs nucleic acid single linear DNA (synthetic) 388 ATCCTGATGG AAAATAACCT TCGAAGGGTT AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 389 CCTGTTGAAT GCCTCCAGGT TAAC 24 18 base pairs nucleic acid single linear DNA (synthetic) 390 AAAAATCTCG CTCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 391 AGCGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 392 AAAAATCTCA ACCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 393 GTTGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 394 AAAAATCTCG AACCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 395 TTCGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 396 AAAAATCTCC ACCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 397 GTGGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 398 AAAAATCTCA TCCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 399 GATGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 400 AAAAATCTCA TGCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 401 CATGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 402 AAAAATCTCT TCCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 403 GAAGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 404 AAAAATCTCT CCCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 405 GGAGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 406 AAAAATCTCA CCCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 407 GGTGAGATTT TTAAGAAT 18 18 base pairs nucleic acid single linear DNA (synthetic) 408 AAAAATCTCT ACCCATGT 18 18 base pairs nucleic acid single linear DNA (synthetic) 409 GTAGAGATTT TTAAGAAT 18 27 base pairs nucleic acid single linear DNA (synthetic) 410 CTGCCCCTGG CCACGGCCGC AGCTACG 27 24 base pairs nucleic acid single linear DNA (synthetic) 411 ATGGATTGGA TGTCGCGTAG CTGC 24 27 base pairs nucleic acid single linear DNA (synthetic) 412 CTGCCCCTGG CCACGGCCGC AGGTACG 27 24 base pairs nucleic acid single linear DNA (synthetic) 413 ATGGATTGGA TGTCGCGTAC CTGC 24 27 base pairs nucleic acid single linear DNA (synthetic) 414 CTGCCCCTGG CCACGGCCGC AATCACG 27 24 base pairs nucleic acid single linear DNA (synthetic) 415 ATGGATTGGA TGTCGCGTGA TTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 416 GCTCATCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 417 ATGGATTGGA TGAGCCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 418 CAGCATCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 419 ATGGATTGGA TGCTGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 420 CACCATCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 421 ATGGATTGGA TGGTGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 422 AAACATCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 423 ATGGATTGGA TGTTTCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 424 CGAGCTCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 425 ATGGATTGGA GCTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 426 CGAAACCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 427 ATGGATTGGG TTTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 428 CGAGACCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 429 ATGGATTGGG TCTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 430 CGAATCCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 431 ATGGATTGGG ATTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 432 CGAAAACCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 433 ATGGATTGGT TTTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 434 CGAATGCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 435 ATGGATTGGC ATTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 436 CGATTCCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 437 ATGGATTGGG AATCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 438 CGATCCCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 439 ATGGATTGGG GATCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 440 CGATGGCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 441 ATGGATTGGC CATCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 442 CGATACCCAA TCCATATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 443 ATGGATTGGG TATCGCGTGG GTGC 24 34 base pairs nucleic acid single linear DNA (synthetic) 444 CATCCAATCC AAATCAAGGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 445 AATTCATTCC AGTCACCGTC CTTGATTTGG ATTGGATGTC GCGT 44 34 base pairs nucleic acid single linear DNA (synthetic) 446 CATCCAATCG AAATCAAGGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 447 AATTCATTCC AGTCACCGTC CTTGATTTCG ATTGGATGTC GCGT 44 34 base pairs nucleic acid single linear DNA (synthetic) 448 CATCCAATCA TGATCAAGGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 449 AATTCATTCC AGTCACCGTC CTTGATCATG ATTGGATGTC GCGT 44 34 base pairs nucleic acid single linear DNA (synthetic) 450 CATCCAATCT TCATCAAGGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 451 AATTCATTCC AGTCACCGTC CTTGATGAAG ATTGGATGTC GCGT 44 34 base pairs nucleic acid single linear DNA (synthetic) 452 CATCCAATCT CCATCAAGGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 453 AATTCATTCC AGTCACCGTC CTTGATGGAG ATTGGATGTC GCGT 44 34 base pairs nucleic acid single linear DNA (synthetic) 454 CATCCAATCG TAATCAAGGA CGGTGACTGG AATG 34 44 base pairs nucleic acid single linear DNA (synthetic) 455 AATTCATTCC AGTCACCGTC CTTGATTACG ATTGGATGTC GCGT 44 21 base pairs nucleic acid single linear DNA (synthetic) 456 CGACATCCAA TCCGTATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 457 ACGGATTGGA TGTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 458 CGACATCCAA TCAAAATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 459 TTTGATTGGA TGTCGCGTGG GTGC 24 21 base pairs nucleic acid single linear DNA (synthetic) 460 CGACATCCAA TCTACATCAA G 21 24 base pairs nucleic acid single linear DNA (synthetic) 461 GTAGATTGGA TGTCGCGTGG GTGC 24 16 base pairs nucleic acid single linear DNA (synthetic) 462 GCTGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 463 AATTCATTCC AGTCACCAGC CTTGAT 26 16 base pairs nucleic acid single linear DNA (synthetic) 464 AACGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 465 AATTCATTCC AGTCACCGTT CTTGAT 26 16 base pairs nucleic acid single linear DNA (synthetic) 466 GAAGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 467 AATTCATTCC AGTCACCTTC CTTGAT 26 16 base pairs nucleic acid single linear DNA (synthetic) 468 GGTGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 469 AATTCATTCC AGTCACCACC CTTGAT 26 16 base pairs nucleic acid single linear DNA (synthetic) 470 ATCGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 471 AATTCATTCC AGTCACCGAT CTTGAT 26 16 base pairs nucleic acid single linear DNA (synthetic) 472 CTGGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 473 AATTCATTCC AGTCACCCAG CTTGAT 26 16 base pairs nucleic acid single linear DNA (synthetic) 474 TTCGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 475 AATTCATTCC AGTCACCGAA CTTGAT 26 16 base pairs nucleic acid single linear DNA (synthetic) 476 TCCGGTGACT GGAATG 16 26 base pairs nucleic acid single linear DNA (synthetic) 477 AATTCATTCC AGTCACCGGA CTTGAT 26 32 base pairs nucleic acid single linear DNA (synthetic) 478 AATTCGCTAG GAAACTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 479 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTAGCG 37 32 base pairs nucleic acid single linear DNA (synthetic) 480 AATTCCAGAG GAAACTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 481 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCTGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 482 AATTCCACAG GAAACTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 483 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGTGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 484 AATTCTCCAG GAAACTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 485 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGGAG 37 32 base pairs nucleic acid single linear DNA (synthetic) 486 AATTCCGGAG GCGTCTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 487 CTCAAGGGTT TTCAGATAGA ACGTCAGACG CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 488 AATTCCGGAG GGAACTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 489 CTCAAGGGTT TTCAGATAGA ACGTCAGTTC CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 490 AATTCCGGAG GCACCTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 491 CTCAAGGGTT TTCAGATAGA ACGTCAGGTG CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 492 AATTCCGGAG GATCCTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 493 CTCAAGGGTT TTCAGATAGA ACGTCAGGAT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 494 AATTCCGGAG GTCCCTGACG TTCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 495 CTCAAGGGTT TTCAGATAGA ACGTCAGGGA CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 496 AATTCCGGAG GAAACTGACG GACTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 497 CTCAAGGGTT TTCAGATAGT CCGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 498 AATTCCGGAG GAAACTGACG ATCTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 499 CTCAAGGGTT TTCAGATAGA TCGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 500 AATTCCGGAG GAAACTGACG CTGTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 501 CTCAAGGGTT TTCAGATACA GCGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 502 AATTCCGGAG GAAACTGACG AAATATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 503 CTCAAGGGTT TTCAGATATT TCGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 504 AATTCCGGAG GAAACTGACG GTTTATCTGA AA 32 37 base pairs nucleic acid single linear DNA (synthetic) 505 CTCAAGGGTT TTCAGATAAA CCGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 506 AATTCCGGAG GAAACTGACG TTCTATCTGG CT 32 37 base pairs nucleic acid single linear DNA (synthetic) 507 CTCAAGGGTA GCCAGATAGA ACGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 508 AATTCCGGAG GAAACTGACG TTCTATCTGC GT 32 37 base pairs nucleic acid single linear DNA (synthetic) 509 CTCAAGGGTA CGCAGATAGA ACGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 510 AATTCCGGAG GAAACTGACG TTCTATCTGA AC 32 37 base pairs nucleic acid single linear DNA (synthetic) 511 CTCAAGGGTG TTCAGATAGA ACGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 512 AATTCCGGAG GAAACTGACG TTCTATCTGC AG 32 37 base pairs nucleic acid single linear DNA (synthetic) 513 CTCAAGGGTC TGCAGATAGA ACGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 514 AATTCCGGAG GAAACTGACG TTCTATCTGC AC 32 37 base pairs nucleic acid single linear DNA (synthetic) 515 CTCAAGGGTG TGCAGATAGA ACGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 516 AATTCCGGAG GAAACTGACG TTCTATCTGA TG 32 37 base pairs nucleic acid single linear DNA (synthetic) 517 CTCAAGGGTC ATCAGATAGA ACGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 518 AATTCCGGAG GAAACTGACG TTCTATCTGT TC 32 37 base pairs nucleic acid single linear DNA (synthetic) 519 CTCAAGGGTG AACAGATAGA ACGTCAGTTT CCTCCGG 37 32 base pairs nucleic acid single linear DNA (synthetic) 520 AATTCCGGAG GAAACTGACG TTCTATCTGT AC 32 37 base pairs nucleic acid single linear DNA (synthetic) 521 CTCAAGGGTG TACAGATAGA ACGTCAGTTT CCTCCGG 37 40 base pairs nucleic acid single linear DNA (synthetic) 522 CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA 40 36 base pairs nucleic acid single linear DNA (synthetic) 523 CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC 36 27 base pairs nucleic acid single linear DNA (synthetic) 524 AACAACCTCA ATGGGGAAGA CCAAGAT 27 45 base pairs nucleic acid single linear DNA (synthetic) 525 CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC 45 36 base pairs nucleic acid single linear DNA (synthetic) 526 GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG 36 18 base pairs nucleic acid single linear DNA (synthetic) 527 ATCTTGGTCT TCCCCATT 18 36 base pairs nucleic acid single linear DNA (synthetic) 528 ATCCTGATGG AAAATAACCT TCGAAGGCCA AACCTG 36 24 base pairs nucleic acid single linear DNA (synthetic) 529 GAGGCATTCA ACAGGGCTGT CAAG 24 15 base pairs nucleic acid single linear DNA (synthetic) 530 AGTTTACAGA ATGCA 15 27 base pairs nucleic acid single linear DNA (synthetic) 531 CCTTCGAAGG TTATTTTCCA TCAGGAT 27 24 base pairs nucleic acid single linear DNA (synthetic) 532 CCTGTTGAAT GCCTCCAGGT TTGG 24 20 base pairs nucleic acid single linear DNA (synthetic) 533 TTCTGTAAAC TCTTGACAGC 20 21 base pairs nucleic acid single linear DNA (synthetic) 534 TCAGCAATTG AGAGCATTCT T 21 18 base pairs nucleic acid single linear DNA (synthetic) 535 AAAAATCTCC TGCCATGT 18 48 base pairs nucleic acid single linear DNA (synthetic) 536 CTGCCCCTGG CCACGGCCGC ACCCACGCGA CATCCAATCC ATATCAAG 48 27 base pairs nucleic acid single linear DNA (synthetic) 537 CTGCCCCTGG CCACGGCCGC ACCCACG 27 21 base pairs nucleic acid single linear DNA (synthetic) 538 CGACATCCAA TCCATATCAA G 21 16 base pairs nucleic acid single linear DNA (synthetic) 539 GACGGTGACT GGAATG 16 19 base pairs nucleic acid single linear DNA (synthetic) 540 GCTCTCAATT GCTGATGCA 19 18 base pairs nucleic acid single linear DNA (synthetic) 541 CAGGAGATTT TTAAGAAT 18 48 base pairs nucleic acid single linear DNA (synthetic) 542 ATGGATTGGA TGTCGCGTGG GTGCGGCCGT GGCCAGGGGC AGACATGG 48 24 base pairs nucleic acid single linear DNA (synthetic) 543 GGCCGTGGCC AGGGGCAGAC ATGG 24 24 base pairs nucleic acid single linear DNA (synthetic) 544 ATGGATTGGA TGTCGCGTGG GTGC 24 26 base pairs nucleic acid single linear DNA (synthetic) 545 AATTCATTCC AGTCACCGTC CTTGAT 26 32 base pairs nucleic acid single linear DNA (synthetic) 546 AATTCCGGAG GAAACTGACG TTCTATCTGA AA 32 32 base pairs nucleic acid single linear DNA (synthetic) 547 ACCCTTGAGA ATGCGCAGGC TCAACAGTAA TA 32 37 base pairs nucleic acid single linear DNA (synthetic) 548 CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCCGG 37 27 base pairs nucleic acid single linear DNA (synthetic) 549 AGCTTATTAC TGTTGAGCCT GCGCATT 27 

What is claimed is:
 1. A method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and (c) harvesting said cultured cells.
 2. A method for selective ex-vivo expansion of stem cells, comprising the steps of, (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or, Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cyg, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; and (c) harvesting said cultured cells.
 3. A method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein; Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and (c) harvesting said cultured cells.
 4. A method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:130; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His, Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val, Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and (c) harvesting said cultured cells.
 5. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
 6. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at poSition 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu Tyr; His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 49 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
 7. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells from the patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3 and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
 8. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:30; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
 9. The method according to claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein said modified human interleukin-3 polypeptide has at least five times greater cell proliferative activity than native human interleukin-3.
 10. The method according to claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein said modified human interleukin-3 polypeptide has at least ten times greater cell proliferative activity than native human interleukin-3.
 11. The method according to claim 2 or 6, wherein said mutant human interleukin-3 polypeptide is selected from the group consisting of: (a) a polypeptide having an amino acid sequence of (i) residues 3-113 of SEQ ID NO:66; (ii) residues 3-113 of SEQ ID NO:67; (iii) residues 3-113 of SEQ ID NO:69; or (iv) SEQ ID NO:218; and (b) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (a).
 12. A method for selective ex-vivo expansion of stem cells, consisting of the steps of; (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and (c) harvesting said cultured cells.
 13. A method for selective ex-vivo expansion of stem cells, consisting of the steps of; (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at PoSition 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; and (c) harvesting said cultured cells.
 14. A method for selective ex-vivo expansion of stem cells, consisting of the steps of; (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and (c) harvesting said cultured cells.
 15. A method for selective ex-vivo expansion of stem cells, consisting of the steps of; (a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:130; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and (c) harvesting said cultured cells.
 16. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of; (a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and (e) transplanting said cultured cells into said patient.
 17. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of; (a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu, Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
 18. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of; (a) removing stem cells from the patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val; Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3 and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
 19. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of; (a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:130; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val, Trp or Ser; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
 20. The method according to claim 12, 13, 14, 15, 16, 17, 18, or 19, wherein said modified human interleukin-3 polypeptide has at least five times greater cell proliferative activity than native human interleukin-3.
 21. The method according to claim 12, 13, 14, 15, 16, 17, 18, or 19 wherein said modified human interleukin-3 polypeptide has at least ten times greater cell proliferative activity than native human interleukin-3.
 22. The method according to claim 13 or 17, wherein said mutant human interleukin-3 polypeptide is selected from the group consisting of: (a) a polypeptide having an amino acid sequence of (i) residues 3-113 of SEQ ID NO:66; (ii) residues 3-113 of SEQ ID NO:67; (iii) residues 3-113 of SEQ ID NO:69; or (iv) SEQ ID NO:218; and (b) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (a). 